Anterograde Axonal Transport Research Articles

Progressive Optic Neuropathy in Hydrocephalic Ccdc13 Mutant Mice Caused by Impaired Axoplasmic Transport at the Optic Nerve Head.

Optic nerve head (ONH) atrophy is frequently associated with hydrocephalic conditions. Cerebrospinal fluid (CSF)-containing meninges form a subarachnoid space that terminates at the ONH, which physically impacts it. This study aims to characterize optic neuropathy in congenital hydrocephalic mice with genetic disruption of the Ccdc13 gene. The ccdc13 germline knockout mice were generated. The hydrocephalus phenotype and subarachnoid space surrounding the optic nerve were evaluated using routine histology and Evans blue stain. Optic neuropathy was examined with immunohistochemistry and transmission electron microscopy (TEM). Axon transport was indicated by cholera toxin subunit B (CTB) fluorescence conjugate. Retinal function was evaluated by electroretinography (ERG), and Ccdc13 expression was revealed by a knock-in Gfp reporter. Ccdc13 mutant mice manifested hydrocephalus at birth. ONH displacement, or negative cupping, and enlarged subarachnoid space at the optic terminus occurred as early as 1 month after birth. Intraocular pressure (IOP) was normal. Optic neuropathy was first observed at the ONH, followed by a distal-to-proximal progression of optic nerve pathology indicated by alteration of axonal ultrastructure and deposition of unphosphorylated neurofilament heavy chain. Anterograde axonal transport was also hampered. Retinal ganglion cell (RGC) function was compromised as early as postnatal day 21 (P21), along with reduced neurofilament heavy chain expression. Optic neuropathy caused by disruption of Ccd13 was non-cell autonomous, stemming from hydrocephalus with presumed high intracranial pressure (ICP), which physically impacts the ONH by increasing the translaminar pressure gradient. We provided knowledge of optic neuropathy from a congenital mouse model for hydrocephalus. The hydrocephalus in mice could damage the ONH by increasing the translaminar pressure gradient and negative cupping, leading to impairment in axoplasmic transport and RGC pathology. Our findings highlight the importance of the interplay between IOP and ICP in the development of glaucoma.

Impaired axonal transport at the optic nerve head contributes to neurodegeneration in a novel Cre-inducible mouse model of myocilin glaucoma.

Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction, leading to neurodegeneration, is the pathological hallmark of primary open-angle glaucoma (POAG). Impaired axonal transport is an early and critical feature of glaucomatous neurodegeneration. However, a robust mouse model that replicates these human POAG features accurately has been lacking. We report the development and characterization of a novel Cre-inducible mouse model expressing a DsRed-tagged Y437H mutant of human myocilin (Tg.CreMYOCY437H). A single intravitreal injection of HAd5-Cre induced selective MYOC expression in the TM, causing TM dysfunction, reducing outflow facility, and progressively elevating IOP in Tg.CreMYOCY437H mice. Sustained IOP elevation resulted in significant retinal ganglion cell (RGC) loss and progressive axonal degeneration in Cre-induced Tg.CreMYOCY437H mice. Notably, impaired anterograde axonal transport was observed at the optic nerve head before RGC degeneration, independent of age, indicating that impaired axonal transport contributes to RGC degeneration in Tg.CreMYOCY437H mice. In contrast, axonal transport remained intact in ocular hypertensive mice injected with microbeads, despite significant RGC loss. Our findings indicate that Cre-inducible Tg.CreMYOCY437H mice replicate all glaucoma phenotypes, providing an ideal model for studying early events of TM dysfunction and neuronal loss in POAG.

The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation

Mitochondrial dysfunction is a prominent hallmark of Alzheimer's disease (AD). The transcriptional coactivator PPARγ coactivator 1 (PGC-1a) has been identified as a key regulator of mitochondrial biogenesis and function. However, the precise structure/function relationship between PGC-1a and mitochondrial quality control remains incompletely understood. In this study, we investigated the impact of PGC-1a on AD pathology and its underlying mechanisms with a specific focus on mitochondrial axonal transport. Additionally, we generated two PGC-1α mutants by substituting leucine residues at positions 148 and 149 within the LKKLL motif or at positions 209 and 210 within the LLKYL motif with alanine. Subsequently, we examined the effects of these mutants on mutAPP-induced abnormalities in anterograde and retrograde axonal transport, disrupted mitochondrial distribution, and impaired mitophagy.Mutagenesis studies revealed that the LLKYL motif at amino acid position 209–210 within PGC-1α plays an essential role in its interaction with estrogen-related receptors (ERRα), which is necessary for restoring normal mitochondrial anterograde axonal transport, maintaining proper mitochondrial distribution, and ultimately preventing neuronal apoptosis. Furthermore, it was found that the Leu-rich motif at amino acids 209–210 within PGC-1α is crucial for rescuing mutAPP-induced impairment in mitophagy and loss of membrane potential by restoring normal mitochondrial retrograde axonal transport. Conversely, mutation of residues 148 and 149 in the LKKLL motif does not compromise the effectiveness of PGC-1α. These findings provide valuable insights into the molecular determinants governing specificity of action for PGC-1α involved in regulating mutAPP-induced deficits in mitochondrial axonal trafficking. Moreover, they suggest a potential therapeutic target for addressing Alzheimer's disease.

Circadian Vesicle Capture Prepares Clock Neuron Synapses for Daily Phase-Delayed Neuropeptide Release.

Drosophila sLNv clock neurons release the neuropeptide PDF to control circadian rhythms. Strikingly, PDF content in sLNv terminals is rhythmic with a peak in the morning hours prior to the onset of activity-dependent release. Because synaptic PDF accumulation, rather than synaptic release, aligns with the late-night elevations in both sLNv neuron excitability and Ca2+, we explored the dependence of presynaptic neuropeptide accumulation on neuropeptide vesicle transport, electrical activity and the circadian clock. Live imaging reveals that anterograde axonal transport is constant throughout the day and capture of circulating neuropeptide vesicles rhythmically boosts presynaptic neuropeptide content hours prior to release. The late-night surge in vesicle capture, like release, requires electrical activity and results in a large releasable pool of presynaptic vesicles to support the later burst of neuropeptide release. The circadian clock is also required suggesting that it controls the switch from vesicle capture to exocytosis, which are normally coupled activity-dependent processes. This toggling of activity transduction maximizes rhythmic synaptic neuropeptide release needed for robust circadian behavior and resolves the previously puzzling delay in timing of synaptic neuropeptide release relative to changes in sLNv clock neuron physiology.

Kinesin Regulation in the Proximal Axon is Essential for Dendrite-selective Transport.

Neurons are polarized and typically extend multiple dendrites and one axon. To maintain polarity, vesicles carrying dendritic proteins are arrested upon entering the axon. To determine whether kinesin regulation is required for terminating anterograde axonal transport, we overexpressed the dendrite-selective kinesin KIF13A. This caused mistargeting of dendrite-selective vesicles to the axon and a loss of dendritic polarity. Polarity was not disrupted if the kinase MARK2/Par1b was coexpressed. MARK2/Par1b is concentrated in the proximal axon, where it maintains dendritic polarity-likely by phosphorylating S1371 of KIF13A, which lies in a canonical 14-3-3 binding motif. We probed for interactions of KIF13A with 14-3-3 isoforms and found that 14-3-3β and 14-3-3ζ bound KIF13A. Disruption of MARK2 or 14-3-3 activity by small molecule inhibitors caused a loss of dendritic polarity. These data show that kinesin regulation is integral for dendrite-selective transport. We propose a new model in which KIF13A that moves dendrite-selective vesicles in the proximal axon is phosphorylated by MARK2. Phosphorylated KIF13A is then recognized by 14-3-3, which causes dissociation of KIF13A from the vesicle and termination of transport. These findings define a new paradigm for the regulation of vesicle transport by localized kinesin tail phosphorylation, to restrict dendrite-selective vesicles from entering the axon.

Early Chronic Fluoxetine Treatment of Ts65Dn Mice Rescues Synaptic Vesicular Deficits and Prevents Aberrant Proteomic Alterations.

Down syndrome (DS) is the most common form of inherited intellectual disability caused by trisomy of chromosome 21, presenting with intellectual impairment, craniofacial abnormalities, cardiac defects, and gastrointestinal disorders. The Ts65Dn mouse model replicates many abnormalities of DS. We hypothesized that investigation of the cerebral cortex of fluoxetine-treated trisomic mice may provide proteomic signatures that identify therapeutic targets for DS. Subcellular fractionation of synaptosomes from cerebral cortices of age- and brain-area-matched samples from fluoxetine-treated vs. water-treated trisomic and euploid male mice were subjected to HPLC-tandem mass spectrometry. Analysis of the data revealed enrichment of trisomic risk genes that participate in regulation of synaptic vesicular traffic, pre-synaptic and post-synaptic development, and mitochondrial energy pathways during early brain development. Proteomic analysis of trisomic synaptic fractions revealed significant downregulation of proteins involved in synaptic vesicular traffic, including vesicular endocytosis (CLTA, CLTB, CLTC), synaptic assembly and maturation (EXOC1, EXOC3, EXOC8), anterograde axonal transport (EXOC1), neurotransmitter transport to PSD (SACM1L), endosomal-lysosomal acidification (ROGDI, DMXL2), and synaptic signaling (NRXN1, HIP1, ITSN1, YWHAG). Additionally, trisomic proteomes revealed upregulation of several trafficking proteins, involved in vesicular exocytosis (Rab5B), synapse elimination (UBE3A), scission of endocytosis (DBN1), transport of ER in dendritic spines (MYO5A), presynaptic activity-dependent bulk endocytosis (FMR1), and NMDA receptor activity (GRIN2A). Chronic fluoxetine treatment of Ts65Dn mice rescued synaptic vesicular abnormalities and prevented abnormal proteomic changes in adult Ts65Dn mice, pointing to therapeutic targets for potential treatment of DS.

RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors.

Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adaptor MADD, potentially preventing the formation of the RAB3A-MADD-KIF1A/1Bβ complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD.

Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport.

Mitochondria transport is crucial for axonal mitochondria distribution and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 binding to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1, and metaxin2. We conclude that transport complexes containing kinesin-1 and RIC-7 polarize at the leading edge of mitochondria and are required for anterograde axonal transport in C. elegans.

Equine grass sickness is associated with major abnormalities in the ultrastructure of skeletal neuromuscular junctions.

Equine grass sickness (EGS) is a frequently fatal multisystem neuropathy of equids. The aetiology is unknown; proposed causes include toxicoinfection with Clostridium botulinum and a mycotoxicosis. The effect of EGS on the organisation and structural integrity of the skeletal neuromuscular junction (NMJ), the target of botulinum neurotoxins (BoNTs), is unknown. To compare the organisation and structural integrity of skeletal NMJs from EGS horses, control horses and one horse with a presumptive diagnosis of botulism. Blinded, retrospective case control. NMJs in samples of diaphragm or intercostal muscle from six EGS horses, three control horses and one equine botulism case were compared using electron microscopy, morphometry and confocal light microscopy. A significantly higher percentage of EGS NMJs had abnormal morphology (EGS 72.2%, 95% CI 55.6-84.4; Controls 6.9%, 1.7-23.8; OR 35.1, 8.47-244.8; p < 0.001). EGS NMJs had a significantly lower mean volume fraction occupied by synaptic vesicles (SVs) (EGS 18.7%, 12.6-28.0; Controls 36.3%, 20.8-63.4; p = 0.024). EGS NMJs had evidence of accelerated SV exocytosis and SV depletion, accumulation of neurofilament-like material in terminal boutons and/or bouton degeneration. NMJs from the botulism horse had dense packing of SVs towards the presynaptic membrane active zone, consistent with BoNT intoxication, but had absence of the abnormalities identified in EGS NMJs. Group sizes were limited by difficulties obtaining suitably processed samples. Ages of control and EGS horses differed. Botulism was diagnosed based on clinical and post mortem findings. EGS is associated with major changes in skeletal NMJ ultrastructure that are inconsistent with the effects of BoNTs. SV depletion may reflect increased exocytosis coupled with reduced repopulation of SVs via anterograde axonal transport and endocytosis, consistent with the action of an excitatory presynaptic toxin and/or neurotransmitter reuptake inhibitor. Skeletal NMJs represent a previously unrecognised target for the toxin that causes EGS.

Altered anterograde axonal transport of mitochondria in cultured striatal neurons of a knock-in mouse model of Huntington's disease

Huntington's disease (HD) is a progressive genetic neurodegenerative disease caused by an abnormal expansion of a cytosine-adenine-guanine trinucleotide repeat in the huntingtin gene. One pathological feature of HD is neuronal loss in the striatum. Despite many efforts, mechanisms underlying neuronal loss in HD striatum remain elusive. It was suggested that the mutant huntingtin protein interacts mitochondrial proteins and causes mitochondrial dysfunction in striatal neurons. However, whether axonal transport of mitochondria is altered in HD striatal neurons remains controversial. Here, we examined axonal transport of single mitochondria labelled with Mito-DsRed2 in cultured striatal neurons of zQ175 knock-in mice (a knock-in mouse model of HD). We observed decreased anterograde axonal transport of proximal mitochondria in HD striatal neurons compared with wild-type (WT) striatal neurons. Decreased anterograde transport in HD striatal neurons was prevented by overexpressing mitochondrial Rho GTPase 1 (Miro1). Our results offer a new insight into mechanisms underlying neuronal loss in the striatum in HD.

Neutral sphingomyelinase inhibition promotes local and network degeneration in vitro and in vivo

BackgroundCell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may contain cargoes that promote survival, neuroprotection, or pathology. Our understanding of pathologic intercellular signaling has been bolstered by disease models using neurons derived from human pluripotent stems cells (hPSC).MethodsHere, we used hPSC-derived retinal ganglion cells (hRGC) and the mouse visual system to investigate the influence of modulating EV generation on intercellular trafficking and cell survival. We probed the impact of EV modulation on cell survival by decreasing the catabolism of sphingomyelin into ceramide through inhibition of neutral sphingomyelinase (nSMase), using GW4869. We assayed for cell survival in vitro by probing for annexin A5, phosphatidylserine, viable mitochondria, and mitochondrial reactive oxygen species. In vivo, we performed intraocular injections of GW4869 and measured RGC and superior colliculus neuron density and RGC anterograde axon transport.ResultsFollowing twenty-four hours of dosing hRGCs with GW4869, we found that inhibition of nSMase decreased ceramide and enhanced GM1 ganglioside accumulation. This inhibition also reduced the density of small EVs, increased the density of large EVs, and enriched the pro-apoptotic protein, annexin A5. Reducing nSMase activity increased hRGC apoptosis initiation due to enhanced density and uptake of apoptotic particles, as identified by the annexin A5 binding phospholipid, phosphatidylserine. We assayed intercellular trafficking of mitochondria by developing a coculture system of GW4869-treated and naïve hRGCs. In treated cells, inhibition of nSMase reduced the number of viable mitochondria, while driving mitochondrial reactive oxygen species not only in treated, but also in naive hRGCs added in coculture. In mice, 20 days following a single intravitreal injection of GW4869, we found a significant loss of RGCs and their axonal recipient neurons in the superior colliculus. This followed a more dramatic reduction in anterograde RGC axon transport to the colliculus.ConclusionOverall, our data suggest that perturbing the physiologic catabolism of sphingomyelin by inhibiting nSMase reorganizes plasma membrane associated sphingolipids, alters the profile of neuron-generated EVs, and promotes neurodegeneration in vitro and in vivo by shifting the balance of pro-survival versus -degenerative EVs.8WXtUmcgzqdrdTPfGd7tbXVideo

Effect of mitochondrial circulation on mitochondrial age density distribution.

Recent publications report that although the mitochondria population in an axon can be quickly replaced by a combination of retrograde and anterograde axonal transport (often within less than 24 hours), the axon contains much older mitochondria. This suggests that not all mitochondria that reach the soma are degraded and that some are recirculating back into the axon. To explain this, we developed a model that simulates mitochondria distribution when a portion of mitochondria that return to the soma are redirected back to the axon rather than being destroyed in somatic lysosomes. Utilizing the developed model, we studied how the percentage of returning mitochondria affects the mean age and age density distributions of mitochondria at different distances from the soma. We also investigated whether turning off the mitochondrial anchoring switch can reduce the mean age of mitochondria. For this purpose, we studied the effect of reducing the value of a parameter that characterizes the probability of mitochondria transition to the stationary (anchored) state. The reduction in mitochondria mean age observed when the anchoring probability is reduced suggests that some injured neurons may be saved if the percentage of stationary mitochondria is decreased. The replacement of possibly damaged stationary mitochondria with newly synthesized ones may restore the energy supply in an injured axon. We also performed a sensitivity study of the mean age of stationary mitochondria to the parameter that determines what portion of mitochondria re-enter the axon and the parameter that determines the probability of mitochondria transition to the stationary state. The sensitivity of the mean age of stationary mitochondria to the mitochondria stopping probability increases linearly with the number of compartments in the axon. High stopping probability in long axons can significantly increase mitochondrial age.

Interferon inhibits the release of herpes simplex virus-1 from the axons of sensory neurons.

Herpes simplex virus-1 (HSV-1) is a human pathogen known to cause cold sores and genital herpes. HSV-1 establishes lifelong infections in our sensory neurons, with no cure or vaccine available. HSV-1 can reactivate sporadically and travel back along sensory nerves, where it can form lesions in the oral and genital mucosa, eye, and skin, or be shed asymptomatically. New treatment options are needed as resistance is emerging to current antiviral therapies. Here, we show that interferons (IFNs) are capable of blocking virus release from nerve endings, potentially stopping HSV-1 transmission into the skin. Furthermore, we show that IFNγ has the potential to have widespread antiviral effects in the neuron and may have additional effects on HSV-1 reactivation. Together, this study identifies new targets for the development of immunotherapies to stop the spread of HSV-1 from the nerves into the skin.

Striatonigral distribution of a fluorescent reporter following intracerebral delivery of genome editors.

Introduction: Targeted gene editing is proposed as a therapeutic approach for numerous disorders, including neurological diseases. As the brain is organized into neural networks, it is critical to understand how anatomically connected structures are affected by genome editing. For example, neurons in the substantia nigra pars compacta (SNpc) project to the striatum, and the striatum contains neurons that project to the substantia nigra pars reticulata (SNpr). Methods: Here, we report the effect of injecting genome editors into the striatum of Ai14 reporter mice, which have a LoxP-flanked stop cassette that prevents expression of the red fluorescent protein tdTomato. Two weeks following intracerebral delivery of either synthetic nanocapsules (NCs) containing CRISPR ribonucleoprotein targeting the tdTomato stop cassette or adeno-associated virus (AAV) vectors expressing Cre recombinase, the brains were collected, and the presence of tdTomato was assessed in both the striatum and SN. Results: TdTomato expression was observed at the injection site in both the NC- and AAV-treated groups and typically colocalized with the neuronal marker NeuN. In the SN, tdTomato-positive fibers were present in the pars reticulata, and SNpr area expressing tdTomato correlated with the size of the striatal genome edited area. Conclusion: These results demonstrate in vivo anterograde axonal transport of reporter gene protein products to the SNpr following neuronal genome editing in the striatum.

Differential modification of the C-terminal tails of different α-tubulins and their importance for microtubule function in vivo.

Microtubules (MTs) are built from α-/β-tubulin dimers and used as tracks by kinesin and dynein motors to transport a variety of cargos, such as mRNAs, proteins, and organelles, within the cell. Tubulins are subjected to several post-translational modifications (PTMs). Glutamylation is one of them, and it is responsible for adding one or more glutamic acid residues as branched peptide chains to the C-terminal tails of both α- and β-tubulin. However, very little is known about the specific modifications found on the different tubulin isotypes in vivo and the role of these PTMs in MT transport and other cellular processes in vivo. In this study, we found that in Drosophila ovaries, glutamylation of α-tubulin isotypes occurred clearly on the C-terminal ends of αTub84B and αTub84D (αTub84B/D). In contrast, the ovarian α-tubulin, αTub67C, is not glutamylated. The C-terminal ends of αTub84B/D are glutamylated at several glutamyl sidechains in various combinations. Drosophila TTLL5 is required for the mono- and poly-glutamylation of ovarian αTub84B/D and with this for the proper localization of glutamylated microtubules. Similarly, the normal distribution of kinesin-1 in the germline relies on TTLL5. Next, two kinesin-1-dependent processes, the precise localization of Staufen and the fast, bidirectional ooplasmic streaming, depend on TTLL5, too, suggesting a causative pathway. In the nervous system, a mutation of TTLL5 that inactivates its enzymatic activity decreases the pausing of anterograde axonal transport of mitochondria. Our results demonstrate in vivo roles of TTLL5 in differential glutamylation of α-tubulins and point to the in vivo importance of α-tubulin glutamylation for cellular functions involving microtubule transport.

Impaired motor unit recovery and maintenance in a knock-in mouse model of ALS-associated Kif5a variant

Kinesin family member 5A (KIF5A) is an essential, neuron-specific microtubule-associated motor protein responsible for the anterograde axonal transport of various cellular cargos. Loss of function variants in the N-terminal, microtubule-binding domain are associated with hereditary spastic paraplegia and hereditary motor neuropathy. These variants result in a loss of the ability of the mutant protein to process along microtubules. Contrastingly, gain of function splice-site variants in the C-terminal, cargo-binding domain of KIF5A are associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease involving death of upper and lower motor neurons, ultimately leading to degradation of the motor unit (MU; an alpha motor neuron and all the myofibers it innervates) and death. These ALS-associated variants result in loss of autoinhibition, increased procession of the mutant protein along microtubules, and altered cargo binding. To study the molecular and cellular consequences of ALS-associated variants in vivo, we introduced the murine homolog of an ALS-associated KIF5A variant into C57BL/6 mice using CRISPR-Cas9 gene editing which produced mutant Kif5a mRNA and protein in neuronal tissues of heterozygous (Kif5a+/c.3005+1G>A; HET) and homozygous (Kif5ac.3005+1G>A/c.3005+1G>A; HOM) mice. HET and HOM mice appeared normal in behavioral and electrophysiological (compound muscle action potential [CMAP] and MU number estimation [MUNE]) outcome measures at one year of age. When subjected to sciatic nerve injury, HET and HOM mice have delayed and incomplete recovery of the MUNE compared to wildtype (WT) mice suggesting an impairment in MU repair. Moreover, aged mutant Kif5a mice (aged two years) had reduced MUNE independent of injury, and exacerbation of the delayed and incomplete recovery after injury compared to aged WT mice. These data suggest that ALS-associated variants may result in an impairment of the MU to respond to biological challenges such as injury and aging, leading to a failure of MU repair and maintenance. In this report, we present the behavioral, electrophysiological and pathological characterization of mice harboring an ALS-associated Kif5a variant to understand the functional consequences of KIF5A C-terminal variants in vivo.

Microfluidic Platforms Promote Polarization of Human-Derived Retinal Ganglion Cells That Model Axonopathy.

Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is particularly vulnerable due to the high bioenergetic demand of action potential generation. Here, we prepared retinal ganglion cells derived from human embryonic stem cells (hRGCs) to probe how axonal stress alters AIS morphology. hRGCs were cultured on coverslips or microfluidic platforms. We assayed AIS specification and morphology by immunolabeling against ankyrin G (ankG), an axon-specific protein, and postsynaptic density 95 (PSD-95), a dendrite-specific protein. Using microfluidic platforms that enable fluidic isolation, we added colchicine to the axon compartment to lesion axons. We verified axonopathy by measuring the anterograde axon transport of cholera toxin subunit B and immunolabeling against cleaved caspase 3 (CC3) and phosphorylated neurofilament H (SMI-34). We determined the influence of axon injury on AIS morphology by immunolabeling samples against ankG and measuring AIS distance from soma and length. Based on measurements of ankG and PSD-95 immunolabeling, microfluidic platforms promote the formation and separation of distinct somatic-dendritic versus axonal compartments in hRGCs compared to coverslip cultures. Chemical lesioning of axons by colchicine reduced hRGC anterograde axon transport, increased varicosity density, and enhanced expression of CC3 and SMI-34. Interestingly, we found that colchicine selectively affected hRGCs with axon-carrying dendrites by reducing AIS distance from somas and increasing length, thus suggesting reduced capacity to maintain excitability. Thus, microfluidic platforms promote polarized hRGCs that enable modeling of axonopathy. Microfluidic platforms may be used to assay compartmentalized degeneration that occurs during glaucoma.

Pseudorabies Virus Mutants Lacking US9 Are Defective in Cytoplasmic Assembly and Sorting of Virus Particles into Axons and Not in Axonal Transport.

Herpes simplex virus (HSV) and varicella zoster virus (VZV) rely on transport of virus particles in neuronal axons to spread from sites of viral latency in sensory ganglia to peripheral tissues then on to other hosts. This process of anterograde axonal transport involves kinesin motors that move virus particles rapidly along microtubules. α-herpesvirus anterograde transport has been extensively studied by characterizing the porcine pseudorabies virus (PRV) and HSV, with most studies focused on two membrane proteins: gE/gI and US9. It was reported that PRV and HSV US9 proteins bind to kinesin motors, promoting tethering of virus particles on the motors, and furthering anterograde transport within axons. Alternatively, other models have argued that HSV and PRV US9 and gE/gI function in the cytoplasm and not in neuronal axons. Specifically, HSV gE/gI and US9 mutants are defective in the assembly of virus particles in the cytoplasm of neurons and the subsequent sorting of virus particles to cell surfaces and into axons. However, PRV US9 and gE/gI mutants have not been characterized for these cytoplasmic defects. We examined neurons infected with PRV mutants, one lacking both gE/gI and US9 and the other lacking just US9, by electron microscopy. Both PRV mutants exhibited similar defects in virus assembly and cytoplasmic sorting of virus particles to cell surfaces. As well, the mutants exhibited reduced quantities of infectious virus in neurons and in cell culture supernatants. We concluded that PRV US9 primarily functions in neurons to promote cytoplasmic steps in anterograde transport.

The spatial-temporal reactive changes of compressed optic nerve in a clinically relevant rabbit model of persistent compressive optic nerve axonopathy

The optic nerve (ON) can get compressed in different diseases. However, the pathological and functional changes occurring in the compressed ON over time under constant compression are still unclear. In the present study, we implanted an artificial tube around the optic nerve of a rabbit to primarily create a clinically relevant persistent compressive optic nerve axonopathy (PCOA). Due to the protuberance on the inner ring of the tube, steady and persistent compressions were maintained. In this model, we investigated the thickness of ganglion cell complex (GCC), retinal ganglion cell (RGC) density, axon density of optic nerve, flash visual evoked potential (FVEP), and anterograde axonal transport at various times in four different groups viz. the no comp, 1/2 comp, 3/4 comp, and crush groups. The GCC thickness, RGC density, and axon density of ON were hierarchically and significantly decreased in 1/2 comp, 3/4 comp, and crush groups. Compared to no comp eyes, the P2 amplitude ratio of FVEP was significantly decreased in 3/4 comp but not in 1/2 comp eyes. Only a portion of the optic nerve lost the ability of anterograde axonal transport in the 1/2 comp group. However, it was evident at 2-wpo and more prominent at 4-wpo in 3/4 comp eyes. This study reveals that the compression only induces the homolateral ON axons impairment and the proportion of the affected axons maintains the same for mild compression for at least three months. Furthermore, an underlying threshold effect highlights that mild compression does not require urgent surgery, while the severe compression warrants immediate surgical intervention.

Mitochondrial transport along RGC axons: Strategies to restore energy homeostasis and visual function

AbstractDeficits in mitochondria anterograde axonal transport is a common feature of neurodegenerative diseases including glaucoma. Whether aberrant expression of components in the transport machinery impinge directly on metabolic stress, neuronal death, and circuit dysfunction, is currently unknown. We used multiphoton microscope live imaging to monitor mitochondrial transport along the axons of retinal ganglion cells (RGC), the neurons that die in glaucoma. We found that high intraocular pressure, the most important risk factor to develop the disease, impairs anterograde movement of mitochondria along RGC axons leading to mitochondria depletion and energy decline. Molecular profiling of motor and adaptor proteins revealed loss of Disrupted in schizophrenia (Disc1) expression in glaucomatous RGC. Adeno‐associated virus (AAV)‐based Disc1 gene delivery rescued anterograde transport and replenished mitochondria in proximal and distal axonal segments. Delivery of a genetically encoded ATP sensor combined with longitudinal live imaging revealed that Disc1 supplementation increased ATP production in stressed neurons. Strikingly, Disc1 gene therapy promoted neuronal survival, reversed abnormal RGC calcium dynamics, and restored light‐evoked retinal responses and visual behaviour. Our study provides proof‐of‐principle that enhancing mitochondrial anterograde transport is an effective strategy to alleviate metabolic stress and glaucomatous neurodegeneration.