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Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term integrity of axons depends on intrinsic mechanisms including axonal transport and extrinsic support from adjacent glial cells. The mechanisms of support provided by myelinating oligodendrocytes to underlying axons are only partly understood. Oligodendrocytes release extracellular vesicles (EVs) with properties of exosomes, which upon delivery to neurons improve neuronal viability in vitro. Here, we show that oligodendroglial exosome secretion is impaired in 2 mouse mutants exhibiting secondary axonal degeneration due to oligodendrocyte-specific gene defects. Wild-type oligodendroglial exosomes support neurons by improving the metabolic state and promoting axonal transport in nutrient-deprived neurons. Mutant oligodendrocytes release fewer exosomes, which share a common signature of underrepresented proteins. Notably, mutant exosomes lack the ability to support nutrient-deprived neurons and to promote axonal transport. Together, these findings indicate that glia-to-neuron exosome transfer promotes neuronal long-term maintenance by facilitating axonal transport, providing a novel mechanistic link between myelin diseases and secondary loss of axonal integrity.

The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23–25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.

Dominant mutations in glycyl-tRNA synthetase (GlyRS) cause a subtype of Charcot-Marie-Tooth neuropathy (CMT2D). Although previous studies have shown that GlyRS mutants aberrantly interact with Nrp1, giving insight into the disease’s specific effects on motor neurons, these cannot explain length-dependent axonal degeneration. Here, we report that GlyRS mutants interact aberrantly with HDAC6 and stimulate its deacetylase activity on α-tubulin. A decrease in α-tubulin acetylation and deficits in axonal transport are observed in mice peripheral nerves prior to disease onset. An HDAC6 inhibitor used to restore α-tubulin acetylation rescues axonal transport deficits and improves motor functions of CMT2D mice. These results link the aberrant GlyRS-HDAC6 interaction to CMT2D pathology and suggest HDAC6 as an effective therapeutic target. Moreover, the HDAC6 interaction differs from Nrp1 interaction among GlyRS mutants and correlates with divergent clinical presentations, indicating the existence of multiple and different mechanisms in CMT2D. Mutations in glycyl-tRNA synthetase (GlyRS) cause Charcot-Marie-Tooth disease, a neuromuscular disorder characterized by axonal degeneration. Here the authors show that mutant GlyRS interacts with histone deacetylase 6, resulting in increased deacetylation of α-tubulin and axonal transport deficits.

Recombinant Adeno-associated virus vectors (rAAV) are widely used for gene delivery and multiple naturally occurring serotypes have been harnessed to target cells in different tissues and organs including the brain. Here, we provide a detailed and quantitative analysis of the transduction profiles of rAAV vectors based on six of the most commonly used serotypes (AAV1, AAV2, AAV5, AAV6, AAV8, AAV9) that allows systematic comparison and selection of the optimal vector for a specific application. In our studies we observed marked differences among serotypes in the efficiency to transduce three different brain regions namely the striatum, hippocampus and neocortex of the mouse. Despite the fact that the analyzed serotypes have the general ability to transduce all major cell types in the brain (neurons, microglia, astrocytes and oligodendrocytes), the expression level of a reporter gene driven from a ubiquitous promoter varies significantly for specific cell type / serotype combinations. For example, rAAV8 is particularly efficient to drive transgene expression in astrocytes while rAAV9 appears well suited for the transduction of cortical neurons. Interestingly, we demonstrate selective retrograde transport of rAAV5 along axons projecting from the ventral part of the entorhinal cortex to the dentate gyrus. Furthermore, we show that self-complementing rAAV can be used to significantly decrease the time required for the onset of transgene expression in the mouse brain.

Axon regeneration in the CNS is inhibited by many extrinsic and intrinsic factors. Because these act in parallel, no single intervention has been sufficient to enable full regeneration of damaged axons in the adult mammalian CNS. In the external environment, NogoA and CSPGs are strongly inhibitory to the regeneration of adult axons. CNS neurons lose intrinsic regenerative ability as they mature: embryonic but not mature neurons can grow axons for long distances when transplanted into the adult CNS, and regeneration fails with maturity in in vitro axotomy models. The causes of this loss of regeneration include partitioning of neurons into axonal and dendritic fields with many growth-related molecules directed specifically to dendrites and excluded from axons, changes in axonal signalling due to changes in expression and localization of receptors and their ligands, changes in local translation of proteins in axons, and changes in cytoskeletal dynamics after injury. Also with neuronal maturation come epigenetic changes in neurons, with many of the transcription factor binding sites that drive axon growth-related genes becoming inaccessible. The overall aim for successful regeneration is to ensure that the right molecules are expressed after axotomy and to arrange for them to be transported to the right place in the neuron, including the damaged axon tip.

Activity-dependent neuroprotective protein (ADNP), essential for brain formation, is a frequent autism spectrum disorder (ASD)-mutated gene. ADNP associates with microtubule end-binding proteins (EBs) through its SxIP motif, to regulate dendritic spine formation and brain plasticity. Here, we reveal SKIP, a novel four-amino-acid peptide representing an EB-binding site, as a replacement therapy in an outbred Adnp-deficient mouse model. We discovered, for the first time, axonal transport deficits in Adnp +/− mice (measured by manganese-enhanced magnetic resonance imaging), with significant male–female differences. RNA sequencing evaluations showed major age, sex and genotype differences. Function enrichment and focus on major gene expression changes further implicated channel/transporter function and the cytoskeleton. In particular, a significant maturation change (1 month-five months) was observed in beta1 tubulin (Tubb1) mRNA, only in Adnp +/+ males, and sex-dependent increase in calcium channel mRNA (Cacna1e) in Adnp +/+ males compared with females. At the protein level, the Adnp +/− mice exhibited impaired hippocampal expression of the calcium channel (voltage-dependent calcium channel, Cacnb1) as well as other key ASD-linked genes including the serotonin transporter (Slc6a4), and the autophagy regulator, BECN1 (Beclin1), in a sex-dependent manner. Intranasal SKIP treatment normalized social memory in 8- to 9-month-old Adnp +/− -treated mice to placebo-control levels, while protecting axonal transport and ameliorating changes in ASD-like gene expression. The control, all d -amino analog D-SKIP, did not mimic SKIP activity. SKIP presents a novel prototype for potential ASD drug development, a prevalent unmet medical need.

KIF1A is a kinesin superfamily motor protein that transports synaptic vesicle precursors in axons. Cargo binding stimulates the dimerization of KIF1A molecules to induce processive movement along microtubules. Mutations in human Kif1a lead to a group of neurodegenerative diseases called KIF1A-associated neuronal disorder (KAND). KAND mutations are mostly de novo and autosomal dominant; however, it is unknown if the function of wild-type KIF1A motors is inhibited by heterodimerization with mutated KIF1A. Here, we have established Caenorhabditis elegans models for KAND using CRISPR-Cas9 technology and analyzed the effects of human KIF1A mutation on axonal transport. In our C. elegans models, both heterozygotes and homozygotes exhibited reduced axonal transport. Suppressor screening using the disease model identified a mutation that recovers the motor activity of mutated human KIF1A. In addition, we developed in vitro assays to analyze the motility of heterodimeric motors composed of wild-type and mutant KIF1A. We find that mutant KIF1A significantly impaired the motility of heterodimeric motors. Our data provide insight into the molecular mechanism underlying the dominant nature of de novo KAND mutations.

Inherited peripheral neuropathies are a genetically and phenotypically diverse group of disorders that lead to degeneration of peripheral neurons with resulting sensory and motor dysfunction. Genetic neuropathies that primarily cause axonal degeneration, as opposed to demyelination, are most often classified as Charcot-Marie-Tooth disease type 2 (CMT2) and are the focus of this review. Gene identification efforts over the past three decades have dramatically expanded the genetic landscape of CMT and revealed several common pathological mechanisms among various forms of the disease. In some cases, identification of the precise genetic defect and/or the downstream pathological consequences of disease mutations have yielded promising therapeutic opportunities. In this review, we discuss evidence for pathogenic overlap among multiple forms of inherited neuropathy, highlighting genetic defects in axonal transport, mitochondrial dynamics, organelle-organelle contacts, and local axonal protein translation as recurrent pathological processes in inherited axonal neuropathies. We also discuss how these insights have informed emerging treatment strategies, including specific approaches for single forms of neuropathy, as well as more general approaches that have the potential to treat multiple types of neuropathy. Such therapeutic opportunities, made possible by improved understanding of molecular and cellular pathogenesis and advances in gene therapy technologies, herald a new and exciting phase in inherited peripheral neuropathy.

KIF1A is a kinesin family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules (MTs). In humans, more than 10 point mutations in KIF1A are associated with the motor neuron disease hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus a cogent molecular explanation for how KIF1A mutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found that KIF1A mutations associated with the hereditary SPG lead to hyperactivation of KIF1A motility. Introduction of the corresponding mutations into the Caenorhabditis elegans KIF1A homolog unc-104 revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyperactivation of kinesin motor activity, rather than its loss of function, is a cause of motor neuron disease in humans.

A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses.