Disruption of axonal transport in neurodegeneration

Axonal transport is a highly regulated process that can be modified by adaptors, by phosphorylation of motor proteins and their regulators, by posttranslational modification of microtubules, and by organelle-specific interactions. Both kinesin and dynein are autoinhibited at baseline and require activation to traffic along microtubules. Kinesin autoinhibition occurs by folding of the KHC tail to block the KHC motor from binding to microtubules (1). Dynein, on the other hand, is autoinhibited via dimerization of the motor domains, forming a structure termed the phi-particle (3). Non-dimerized dynein is in an open form that has a higher affinity for binding to microtubules but requires binding with dynactin and cargo adaptors to activate motor activity, likely by modifying the orientation of motor domains to allow processive movement along microtubules (3).

Adaptors. The best-characterized adaptor is dynactin, a cofactor for dynein-mediated axonal transport. Dynactin is a large, multi-subunit 1.1 MDa complex that interacts with dynein and microtubules and is essential for the initiation and activation of dynein-mediated transport (46). Other adaptors that function in concert with dynactin to activate dynein-mediated transport include bicaudal D proteins (7), the Hook protein family (8, 9), and spindly (10). Certain adaptors, such as TRAK2 (11), Hook3 (12), Jip1 (13), and HAP1 (14), can bind to both kinesin and dynein to regulate their trafficking (1).

Phosphorylation. Phosphotransferase activity regulates motor protein function (Figure 2). For example, the kinase glycogen synthase kinase-3β (GSK3β) phosphorylates KHC to inhibit axonal transport and also phosphorylates KLCs to release cargoes (15, 16). Studies using perfusion of kinases and their inhibitors into squid axoplasm showed that the stress-activated protein kinases c-Jun N-terminal kinase 3 (JNK3) and p38 mitogen-activated protein kinase (p38 MAPK) directly phosphorylate KHCs to inhibit anterograde transport (17, 18). Additionally, perfusion of active casein kinase 2 (CK2) in the squid axoplasm reduced bidirectional axonal transport velocities (18).

Regulation of axonal transport.Figure 2

Regulation of axonal transport. Axonal transport is regulated via phosphotransferase activity. The kinases GSK3β and casein kinase 2 (CK2) inhibit anterograde axonal transport via phosphorylation of kinesin light chains, while the kinases JNK and p38 MAPK (p38) inhibit anterograde axonal transport via phosphorylation of kinesin heavy chains. GSK3β phosphorylates dynein intermediate chains to inhibit retrograde axonal transport, while CK1 phosphorylates dynein intermediate chains to activate retrograde axonal transport. Inset: Posttranslational modifications of microtubules. Microtubules are composed of α- and β-tubulin. α-Tubulin can be modified via acetylation, detyrosination, and polyglutamylation, while β-tubulin is modified by polyglutamylation.

Dynein-mediated trafficking is also regulated by phosphorylation. Several studies have indicated that the phosphorylation of DICs can alter dynein-mediated trafficking. The kinase casein kinase 1 (CK1) phosphorylates DICs to regulate dynein-dependent transport (19). Additionally, the kinase GSK3β phosphorylates DICs to reduce DIC interaction with the adaptor NDel1, which regulates dynein motility (20). Notably, phosphorylation of DIC reduced the amount of in vitro binding of dynein to dynactin, indicating that phosphorylation may regulate interaction with this important cofactor (21).

Microtubule regulation. Microtubules are cytoskeletal components shaped like hollow tubes. They are composed of α- and β-tubulin, which dimerize and then polymerize into parallel protofilaments to form a microtubule. Microtubules run the entire length of the axon and form the “tracks” along which kinesin and dynein carry cargo, with the plus end oriented toward the distal axon and the minus end oriented toward the cell body. Microtubule function is highly regulated by posttranslational modifications (PTMs), such as detyrosination, polyglutamylation, and acetylation (Figure 2). This Review will focus on the latter two, as they play a role in axonal transport and have been linked to neurodegeneration.

Evidence for the role of polyglutamylation in neurodegeneration comes from Purkinje cell degeneration (pcd) mutant mice, which have neurodegeneration due to loss of function of cytosolic carboxypeptidase 1 (CCP1), a tubulin deglutaminase (22). Increasing microtubule polyglutamylation inhibits axonal transport (2224).

Microtubule acetylation is another important regulator of axonal transport. Microtubules are acetylated by α-tubulin N-acetyltransferase (ATAT) and deacetylated by histone deacetylase 6 (HDAC6) and sirtuin-2 (SIRT2) (25). Acetylation weakens the lateral interactions between protofilaments, thought to confer flexibility and stabilization of microtubules (2628). Reduction of ATAT1 led to a loss of microtubule acetylation and disruption of axonal transport (29, 30). On the other hand, increasing microtubule acetylation via increasing ATAT1 or preventing deacetylation by HDAC6 rescued axonal transport deficits in disease models (see below) (31, 32).

Adaptors selectively regulate organelle transport. Axonal transport regulation is tightly linked to organelle endocytosis, maturation, signaling, and degradation, indicating that axonal transport may play a role as a “hub” for integrating different cellular processes. To achieve specificity in this process, certain adaptors bind to distinct populations of cargoes, as extensively reviewed in ref. 1. For example, endolysosomal trafficking is highly regulated via specific adaptors and Rab GTPases that function as switches. For example, Rab5 controls early endosome trafficking, while Rab7 regulates late endosome maturation, motility, and fusion with lysosomes (33). Endosomal trafficking is mediated by the Hook1/dynein/dynactin complex for early endosomes, and the adaptors RILP, SKIP, and BORC for late endosomes and lysosomes (3437).

A well-characterized population of retrogradely trafficked organelles are signaling endosomes containing neurotrophins and their receptors. Signaling endosomes are initially formed at the synapse with neurotrophin binding to receptors, and then internalized and transported to the soma for neurotrophic signaling. Signaling endosomes are specifically targeted to dynein via interactions with adaptors, including HAP1, which is important for internalization of certain neurotrophins (38); Hook1, which comigrates with signaling endosomes in the distal axon (34); and BICD1, which directs certain neurotrophins to lysosomes for degradation (39).

Lysosome transport in axons is also extensively regulated. A commonly used marker for lysosomes is LAMP1, which also labels autophagosomes and endosome pathway intermediates. A smaller percentage of LAMP1-labeled organelles in axons have acid hydrolase activity, indicating that most degradation likely occurs in or near the soma (40). Lysosomes are anterogradely transported via interactions with ARL8B and SKIP, the same adaptors that regulate anterograde transport of late endosomes, while retrograde transport is driven by the adaptors JIP3 and JIP4 (41). Autophagosome maturation and axonal trafficking are highly interlinked, as autophagosomes are generated in the distal axon and mature into autolysosomes as they are retrogradely transported (42). Autophagosome transport is regulated by the adaptors JIP1, JIP3, HTT, and HAP1 in a sequential manner as autophagosomes mature (13, 43).

Mitochondria trafficking, on the other hand, is bidirectional and is regulated via interactions between the mitochondrial outer membrane protein Miro and the motor adaptors TRAK proteins and metaxins (11, 44). Synaptic vesicle precursors are primarily trafficked anterogradely from the soma to the synapse, where they are released as mature synaptic vesicles, and are regulated via the KIF1A adaptor MADD, which activates the Rab3 GTPase on synaptic vesicles (45). Finally, dense core vesicles, which anterogradely transport neuropeptides and hormones and then undergo retrograde transport for recycling, are regulated by Arl8, Hook3, and the tyrosine phosphatase PTPN21 (45, 46).

Axonal transport and injury signaling. A key discovery in axonal degeneration research is the SARM1 pathway, an enzymatic pathway that uses sterile α and Toll/interleukin-1 receptor motif–containing protein 1 (SARM1) as a sensor of nicotinamide adenine dinucleotide (NAD) and its upstream activator nicotinamide mononucleotide (NMN) to drive programmed axon degeneration via NAD degradation. A key regulator of this pathway is nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), which converts NMN to NAD. NMNAT2 is synthesized in the cell body and transported into the axon. In the event of an axonal injury leading to disruption of NMNAT transport, NMN levels rise while NAD levels decrease in the axon, which activates SARM1 to induce programmed axonal degeneration (47, 48). In this way, continued anterograde axonal transport of NMNAT2 promotes axonal survival.

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