Swedish Alzheimer's disease variant perturbs activity of retrograde molecular motors and causes widespread derangement of axonal transport pathways.

Experimental studies in flies, mice, and humans suggest a significant role of impaired axonal transport in the pathogenesis of Alzheimer's disease (AD). The mechanisms underlying these impairments in axonal transport, however, remain poorly understood. Here we report that the Swedish familial AD mutation causes a standstill of the amyloid precursor protein (APP) in the axons at the expense of its reduced anterograde transport. The standstill reflects the perturbed directionality of the axonal transport of APP, which spends significantly more time traveling in the retrograde direction. This ineffective movement is accompanied by an enhanced association of dynactin-1 with APP, which suggests that reduced anterograde transport of APP is the result of enhanced activation of the retrograde molecular motor dynein by dynactin-1. The impact of the Swedish mutation on axonal transport is not limited to the APP vesicles since it also reverses the directionality of a subset of early endosomes, which become enlarged and aberrantly accumulate in distal locations. In addition, it also reduces the trafficking of lysosomes due to their less effective retrograde movement. Altogether, our experiments suggest a pivotal involvement of retrograde molecular motors and transport in the mechanisms underlying impaired axonal transport in AD and reveal significantly more widespread derangement of axonal transport pathways in the pathogenesis of AD.

In Vivo Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae.

Cargo transport by molecular motors along microtubules is essential for the function of eukaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals (nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and to the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈90 µm × 60 µm size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces the nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having a nonlinear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated with disease models.

A stop or go switch: glycogen synthase kinase 3ß phosphorylation of the kinesin 1 motor domain at Ser314 halts motility without detaching from microtubules.

It is more than 25 years since the discovery that kinesin 1 is phosphorylated by several protein kinases. However, fundamental questions still remain as to how specific protein kinase(s) contribute to particular motor functions under physiological conditions. Because, within an whole organism, kinase cascades display considerable crosstalk and play multiple roles in cell homeostasis, deciphering which kinase(s) is/are involved in a particular process has been challenging. Previously, we found that GSK3ß plays a role in motor function. Here, we report that a particular site on kinesin 1 motor domain (KHC), S314, is phosphorylated by GSK3ß in vivo. The GSK3ß-phosphomimetic-KHCS314D stalled kinesin 1 motility without dissociating from microtubules, indicating that constitutive GSK3ß phosphorylation of the motor domain acts as a STOP. In contrast, uncoordinated mitochondrial motility was observed in CRISPR/Cas9-GSK3ß non-phosphorylatable-KHCS314A Drosophila larval axons, owing to decreased kinesin 1 attachment to microtubules and/or membranes, and reduced ATPase activity. Together, we propose that GSK3ß phosphorylation fine-tunes kinesin 1 movement in vivo via differential phosphorylation, unraveling the complex in vivo regulatory mechanisms that exist during axonal motility of cargos attached to multiple kinesin 1 and dynein motors.

The glycine arginine-rich domain of the RNA-binding protein nucleolin regulates its subcellular localization.

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway.

Autophagy is a degradative pathway required to maintain homeostasis. Neuronal autophagosomes form constitutively at the axon terminal and mature via lysosomal fusion during dynein-mediated transport to the soma. How the dynein-autophagosome interaction is regulated is unknown. Here, we identify multiple dynein effectors on autophagosomes as they transit along the axons of primary neurons. In the distal axon, JIP1 initiates autophagosomal transport. Autophagosomes in the mid-axon require HAP1 and Huntingtin. We find that HAP1 is a dynein activator, binding the dynein-dynactin complex via canonical and noncanonical interactions. JIP3 is on most axonal autophagosomes, but specifically regulates the transport of mature autolysosomes. Inhibiting autophagosomal transport disrupts maturation, and inhibiting autophagosomal maturation perturbs the association and function of dynein effectors; thus, maturation and transport are tightly linked. These results reveal a novel maturation-based dynein effector handoff on neuronal autophagosomes that is key to motility, cargo degradation, and the maintenance of axonal health.

C9orf72 ALS-FTD: recent evidence for dysregulation of the autophagy-lysosome pathway at multiple levels.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two clinically distinct classes of neurodegenerative disorders. Yet, they share a range of genetic, cellular, and molecular features. Hexanucleotide repeat expansions (HREs) in the C9orf72 gene and the accumulation of toxic protein aggregates in the nervous systems of the affected individuals are among such common features. Though the mechanisms by which HREs cause toxicity is not clear, the toxic gain of function due to transcribed HRE RNA or dipeptide repeat proteins (DPRs) produced by repeat-associated non-AUG translation together with a reduction in C9orf72 expression are proposed as the contributing factors for disease pathogenesis in ALS and FTD. In addition, several recent studies point toward alterations in protein homeostasis as one of the root causes of the disease pathogenesis. In this review, we discuss the effects of the C9orf72 HRE in the autophagy-lysosome pathway based on various recent findings. We suggest that dysfunction of the autophagy-lysosome pathway synergizes with toxicity from C9orf72 repeat RNA and DPRs to drive disease pathogenesis.Abbreviation: ALP: autophagy-lysosome pathway; ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ASO: antisense oligonucleotide; C9orf72: C9orf72-SMCR8 complex subunit; DENN: differentially expressed in normal and neoplastic cells; DPR: dipeptide repeat protein; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; ER: endoplasmic reticulum; FTD: frontotemporal dementia; GAP: GTPase-activating protein; GEF: guanine nucleotide exchange factor; HRE: hexanucleotide repeat expansion; iPSC: induced pluripotent stem cell; ISR: integrated stress response; M6PR: mannose-6-phosphate receptor, cation dependent; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: neurodegenerative disorder; RAN: repeat-associated non-ATG; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SLC66A1/PQLC2: solute carrier family 66 member 1; SMCR8: SMCR8-C9orf72 complex subunit; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; WDR41: WD repeat domain 41.

One proline deletion in the fusion peptide of neurotropic mouse hepatitis virus (MHV) restricts retrograde axonal transport and neurodegeneration.

Mouse hepatitis virus (MHV; murine coronavirus) causes meningoencephalitis, myelitis, and optic neuritis followed by axonal loss and demyelination. This murine virus is used as a common model to study acute and chronic virus-induced demyelination in the central nervous system. Studies with recombinant MHV strains that differ in the gene encoding the spike protein have demonstrated that the spike has a role in MHV pathogenesis and retrograde axonal transport. Fusion peptides (FPs) in the spike protein play a key role in MHV pathogenesis. In a previous study of the effect of deleting a single proline residue in the FP of a demyelinating MHV strain, we found that two central, consecutive prolines are important for cell-cell fusion and pathogenesis. The dihedral fluctuation of the FP was shown to be repressed whenever two consecutive prolines were present, in contrast to the presence of a single proline in the chain. Using this proline-deleted MHV strain, here we investigated whether intracranial injection of this strain can induce optic neuritis by retrograde axonal transport from the brain to the retina through the optic nerve. We observed that the proline-deleted recombinant MHV strain is restricted to the optic nerve, is unable to translocate to the retina, and causes only minimal demyelination and no neuronal death. We conclude that an intact proline dyad in the FP of the recombinant demyelinating MHV strain plays a crucial role in translocation of the virus through axons and subsequent neurodegeneration.

Biodistribution of adeno-associated virus type 2 with mutations in the capsid that contribute to heparan sulfate proteoglycan binding.

We compared the phenotypes of three mutant AAV2 viruses containing mutations in arginine amino acids (R585, R588 and R484) previously shown to be involved in AAV2 heparan sulfate binding. The transduction efficiencies of wild type and mutant viruses were determined in the eye, the brain and peripheral organs following subretinal, striatal and intravenous injection, respectively, in mice and rats. We found that each of the three mutants (the single mutant R585A; the double mutant R585, 588A; and the triple mutant R585, 588, 484A) had a unique phenotype compared to wt and each other. R585A was completely defective for transducing peripheral organs via intravenous injection, suggesting that R585A may be useful for targeting peripheral organs by substitution of peptide ligands in the capsid surface. In the brain, all three mutants displayed widespread transduction, with the double mutant R585, 588A displaying the greatest spread and the greatest number of transduced neurons. The double mutant was also extremely efficient for retrograde transport, while the triple mutant was almost completely defective for retrograde transport. This suggested that R484 may be directly involved in interaction with the transport machinery. Finally, the double mutant also displayed improved transduction of the eye compared to wild type and the other mutants.

p110δ PI3-Kinase Inhibition Perturbs APP and TNFα Trafficking, Reduces Plaque Burden, Dampens Neuroinflammation, and Prevents Cognitive Decline in an Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is associated with the cleavage of the amyloid precursor protein (APP) to produce the toxic amyloid-ß (Aß) peptide. Accumulation of Aß, together with the concomitant inflammatory response, ultimately leads to neuronal death and cognitive decline. Despite AD progression being underpinned by both neuronal and immunological components, therapeutic strategies based on dual targeting of these systems remains unexplored. Here, we report that inactivation of the p110δ isoform of phosphoinositide 3-kinase (PI3K) reduces anterograde axonal trafficking of APP in hippocampal neurons and dampens secretion of the inflammatory cytokine tumor necrosis factor-alpha by microglial cells in the familial AD APPswe/PS1ΔE9 (APP/PS1) mouse model. Moreover, APP/PS1 mice with kinase-inactive PI3Kδ (δD910A) had reduced Aß peptides levels and plaques in the brain and an abrogated inflammatory response compared with APP/PS1 littermates. Mechanistic investigations reveal that PI3Kδ inhibition decreases the axonal transport of APP by eliciting the formation of highly elongated tubular-shaped APP-containing carriers, reducing the levels of secreted Aß peptide. Importantly, APP/PS1/δD910A mice exhibited no spatial learning or memory deficits. Our data highlight inhibition of PI3Kδ as a new approach to protect against AD pathology due to its dual action of dampening microglial-dependent neuroinflammation and reducing plaque burden by inhibition of neuronal APP trafficking and processing.SIGNIFICANCE STATEMENT During Alzheimer's disease (AD), the accumulation of the toxic amyloid-ß (Aß) peptide in plaques is associated with a chronic excessive inflammatory response. Uncovering new drug targets that simultaneously reduce both Aß plaque load and neuroinflammation holds therapeutic promise. Using a combination of genetic and pharmacological approaches, we found that the p110δ isoform of phosphoinositide 3-kinase (PI3K) is involved in anterograde trafficking of the amyloid precursor protein in neurons and in the secretion of tumor necrosis factor-alpha from microglial cells. Genetic inactivation of PI3Kδ reduces Aß plaque deposition and abrogates the inflammatory response, resulting in a complete rescue of the life span and spatial memory performance. We conclude that inhibiting PI3Kδ represents a novel therapeutic approach to ameliorate AD pathology by dampening plaque accumulation and microglial-dependent neuroinflammation.

The Carboxyl Terminus of Tegument Protein pUL21 Contributes to Pseudorabies Virus Neuroinvasion.

Following its entry into cells, pseudorabies virus (PRV) utilizes microtubules to deliver its nucleocapsid to the nucleus. Previous studies have shown that PRV VP1/2 is an effector of dynein-mediated capsid transport. However, the mechanism of PRV for recruiting microtubule motor proteins for successful neuroinvasion and neurovirulence is not well understood. Here, we provide evidence that PRV pUL21 is an inner tegument protein. We tested its interaction with the cytoplasmic light chains using a bimolecular fluorescence complementation (BiFC) assay and observed that PRV pUL21 interacts with Roadblock-1. This interaction was confirmed by coimmunoprecipitation (co-IP) assays. We also determined the efficiency of retrograde and anterograde axonal transport of PRV strains in explanted neurons using a microfluidic chamber system and investigated pUL21's contribution to PRV neuroinvasion in vivo Further data showed that the carboxyl terminus of pUL21 is essential for its interaction with Roadblock-1, and this domain contributes to PRV retrograde axonal transport in vitro and in vivo Our findings suggest that the carboxyl terminus of pUL21 contributes to PRV neuroinvasion.IMPORTANCE Herpesviruses are a group of DNA viruses that infect both humans and animals. Alphaherpesviruses are distinguished by their ability to establish latent infection in peripheral neurons. After entering neurons, the herpesvirus capsid interacts with cellular motor proteins and undergoes retrograde transport on axon microtubules. This elaborate process is vital to the herpesvirus lifecycle, but the underlying mechanism remains poorly understood. Here, we determined that pUL21 is an inner tegument protein of pseudorabies virus (PRV) and that it interacts with the cytoplasmic dynein light chain Roadblock-1. We also observed that pUL21 promotes retrograde transport of PRV in neuronal cells. Furthermore, our findings confirm that pUL21 contributes to PRV neuroinvasion in vivo Importantly, the carboxyl terminus of pUL21 is responsible for interaction with Roadblock-1, and this domain contributes to PRV neuroinvasion. This study offers fresh insights into alphaherpesvirus neuroinvasion and the interaction between virus and host during PRV infection.

Insights on UNC-104-dynein/dynactin interactions and their implications on axonal transport in Caenorhabditis elegans.

Bidirectional cargo transport in neurons can be explained by two models: the "tug-of-war model" for short-range transport, in which several kinesin and dynein motors are bound to the same cargo but travel in opposing directions, and by the "motor coordination model" for long-range transport, in which small adaptors or the cargo itself activates or deactivates opposing motors. Direct interactions between the major axonal transporter kinesin-3 UNC-104(KIF1A) and the dynein/dynactin complex remains unknown. In this study, we dissected and evaluated the interaction sites between UNC-104 and dynein as well as between UNC-104 and dynactin using yeast two-hybrid assays. We found that the DYLT-1(Tctex) subunit of dynein binds near the coiled coil 3 (CC3) of UNC-104, and that the DYRB-1(Roadblock) subunit binds near the CC2 region of UNC-104. Regarding dynactin, we specifically revealed strong interactions between DNC-6(p27) and the FHA-CC3 stretch of UNC-104, as well as between the DNC-5(p25) and the CC2-CC3 region of UNC-104. Motility analysis of motors and cargo in the nervous system of Caenorhabditis elegans revealed impaired transport of UNC-104 and synaptic vesicles in dynein and dynactin mutants (or in RNAi knockdown animals). Further, in these mutants UNC-104 clustering along axons was diminished. Interestingly, when dynamic UNC-104 motors enter a stationary UNC-104 cluster their dwelling times are increased in dynein mutants (suggesting that dynein may act as an UNC-104 activator). In summary, we provide novel insights on how UNC-104 interacts with the dynein/dynactin complex and how UNC-104 driven axonal transport depends on dynein/dynactin in C. elegans neurons.

The pUL37 tegument protein guides alpha-herpesvirus retrograde axonal transport to promote neuroinvasion.

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.

Mitochondrial deficits and abnormal mitochondrial retrograde axonal transport play a role in the pathogenesis of mutant Hsp27-induced Charcot Marie Tooth Disease.

Mutations in the small heat shock protein Hsp27, encoded by the HSPB1 gene, have been shown to cause Charcot Marie Tooth Disease type 2 (CMT-2) or distal hereditary motor neuropathy (dHMN). Protein aggregation and axonal transport deficits have been implicated in the disease. In this study, we conducted analysis of bidirectional movements of mitochondria in primary motor neuron axons expressing wild type and mutant Hsp27. We found significantly slower retrograde transport of mitochondria in Ser135Phe, Pro39Leu and Arg140Gly mutant Hsp27 expressing motor neurons than in wild type Hsp27 neurons, although anterograde movement velocities remained normal. Retrograde transport of other important cargoes, such as the p75 neurotrophic factor receptor was minimally altered in mutant Hsp27 neurons, implicating that axonal transport deficits primarily affect mitochondria and the axonal transport machinery itself is less affected. Investigation of mitochondrial function revealed a decrease in mitochondrial membrane potential in mutant Hsp27 expressing motor axons, as well as a reduction in mitochondrial complex 1 activity, increased vulnerability of mitochondria to mitochondrial stressors, leading to elevated superoxide release and reduced mitochondrial glutathione (GSH) levels, although cytosolic GSH remained normal. This mitochondrial redox imbalance in mutant Hsp27 motor neurons is likely to cause low level of oxidative stress, which in turn will contribute to, and indeed may be the underlying cause of the deficits in mitochondrial axonal transport. Together, these findings suggest that the mitochondrial abnormalities in mutant Hsp27-induced neuropathies may be a primary cause of pathology, leading to further deficits in the mitochondrial axonal transport and onset of disease.

Mutant spastin proteins promote deficits in axonal transport through an isoform-specific mechanism involving casein kinase 2 activation.

Mutations of various genes cause hereditary spastic paraplegia (HSP), a neurological disease involving dying-back degeneration of upper motor neurons. From these, mutations in the SPAST gene encoding the microtubule-severing protein spastin account for most HSP cases. Cumulative genetic and experimental evidence suggests that alterations in various intracellular trafficking events, including fast axonal transport (FAT), may contribute to HSP pathogenesis. However, the mechanisms linking SPAST mutations to such deficits remain largely unknown. Experiments presented here using isolated squid axoplasm reveal inhibition of FAT as a common toxic effect elicited by spastin proteins with different HSP mutations, independent of microtubule-binding or severing activity. Mutant spastin proteins produce this toxic effect only when presented as the tissue-specific M1 isoform, not when presented as the ubiquitously-expressed shorter M87 isoform. Biochemical and pharmacological experiments further indicate that the toxic effects of mutant M1 spastins on FAT involve casein kinase 2 (CK2) activation. In mammalian cells, expression of mutant M1 spastins, but not their mutant M87 counterparts, promotes abnormalities in the distribution of intracellular organelles that are correctable by pharmacological CK2 inhibition. Collectively, these results demonstrate isoform-specific toxic effects of mutant M1 spastin on FAT, and identify CK2 as a critical mediator of these effects.

Microtubule stabilising peptides rescue tau phenotypes in-vivo.

The microtubule cytoskeleton is a highly dynamic, filamentous network underpinning cellular structure and function. In Alzheimer's disease, the microtubule cytoskeleton is compromised, leading to neuronal dysfunction and eventually cell death. There are currently no disease-modifying therapies to slow down or halt disease progression. However, microtubule stabilisation is a promising therapeutic strategy that is being explored. We previously investigated the disease-modifying potential of a microtubule-stabilising peptide NAP (NAPVSIPQ) in a well-established Drosophila model of tauopathy characterised by microtubule breakdown and axonal transport deficits. NAP prevented as well as reversed these phenotypes even after they had become established. In this study, we investigate the neuroprotective capabilities of an analogous peptide SAL (SALLRSIPA). We found that SAL mimicked NAP's protective effects, by preventing axonal transport disruption and improving behavioural deficits, suggesting both NAP and SAL may act via a common mechanism. Both peptides contain a putative 'SIP' (Ser-Ile-Pro) domain that is important for interactions with microtubule end-binding proteins. Our data suggests this domain may be central to the microtubule stabilising function of both peptides and the mechanism by which they rescue phenotypes in this model of tauopathy. Our observations support microtubule stabilisation as a promising disease-modifying therapeutic strategy for tauopathies like Alzheimer's disease.

PINK1 and Parkin are genetic modifiers for FUS-induced neurodegeneration.

Dysregulation of Fused in Sarcoma (FUS) gene expression is associated with fronto-temporal lobar degeneration (FTLD), and missense mutations in the FUS gene have been identified in patients affected by amyotrophic lateral sclerosis (ALS). However, molecular and cellular defects underlying FUS proteinopathy remain to be elucidated. Here, we examined whether genes important for mitochondrial quality control play a role in FUS proteinopathy. In our genetic screening, Pink1 and Park genes were identified as modifiers of neurodegeneration phenotypes induced by wild type (Wt) or ALS-associated P525L-mutant human FUS. Down-regulating expression of either Pink1 or Parkin genes ameliorated FUS-induced neurodegeneration phenotypes. The protein levels of PINK1 and Parkin were elevated in cells overexpressing FUS. Remarkably, ubiquitinylation of Miro1 protein, a downstream target of the E3 ligase activity of Parkin, was also increased in cells overexpressing FUS protein. In fly motor neurons expressing FUS, both motility and processivity of mitochondrial axonal transport were reduced by expression of either Wt- or P525L-mutant FUS. Finally, down-regulating PINK1 or Parkin partially rescued the locomotive defects and enhanced the survival rate in transgenic flies expressing FUS. Our data indicate that PINK1 and Parkin play an important role in FUS-induced neurodegeneration. This study has uncovered a previously unknown link between FUS proteinopathy and PINK1/Parkin genes, providing new insights into the pathogenesis of FUS proteinopathy.

A new mode of mitochondrial transport and polarized sorting regulated by Dynein, Milton and Miro.

Intrinsic cell microtubule (MT) polarity, together with molecular motors and adaptor proteins, determines mitochondrial polarized targeting and MT-dependent transport. In polarized cells, such as neurons, mitochondrial mobility and transport require the regulation of kinesin and dynein by two adaptor proteins, Milton and Miro. Recently, we found that dynein heavy chain 64C (Dhc64C) is the primary motor protein for both anterograde and retrograde transport of mitochondria in the Drosophila bristle. In this study, we show that a molecular lesion in the Dhc64C allele that reduced bristle mitochondrial velocity generated a variant that acts as a 'slow' dynein in an MT-gliding assay, indicating that dynein directly regulates mitochondrial transport. We also showed that in milton-RNAi flies, mitochondrial flux into the bristle shaft, but not velocity, was significantly reduced. Surprisingly, mitochondria retrograde flux, but not net velocity, was significantly decreased in miro-RNAi flies. We thus reveal a new mode of mitochondrial sorting in polarized cell growth, whereby bi-directional mitochondrial transport undertaken exclusively by dynein is regulated by Milton in the anterograde direction and by a Miro-dependent switch to the retrograde direction.

Soluble Conformers of Aß and Tau Alter Selective Proteins Governing Axonal Transport.

UNLABELLED: Despite the demonstration that amyloid-ß (Aß) can trigger increased tau phosphorylation and neurofibrillary tangle (NFT) formation in vivo, the molecular link associating Aß and tau pathologies remains ill defined. Here, we observed that exposure of cultured primary neurons to Aß trimers isolated from brain tissue of subjects with Alzheimer's disease led to a specific conformational change of tau detected by the antibody Alz50. A similar association was supported by postmortem human brain analyses. To study the role of Aß trimers in vivo, we created a novel bigenic Tg-Aß+Tau mouse line by crossing Tg2576 (Tg-Aß) and rTg4510 (Tg-Tau) mice. Before neurodegeneration and amyloidosis, apparent Aß trimers were increased by ∼2-fold in 3-month-old Tg-Aß and Tg-Aß+Tau mice compared with younger mice, whereas soluble monomeric Aß levels were unchanged. Under these conditions, the expression of soluble Alz50-tau conformers rose by ∼2.2-fold in the forebrains of Tg-Aß+Tau mice compared with nontransgenic littermates. In parallel, APP accumulated intracellularly, suggestive of a putative dysfunction of anterograde axonal transport. We found that the protein abundance of the kinesin-1 light chain (KLC1) was reduced selectively in vivo and in vitro when soluble Aß trimers/Alz50-tau were present. Importantly, the reduction in KLC1 was prevented by the intraneuronal delivery of Alz50 antibodies. Collectively, our findings reveal that specific soluble conformers of Aß and tau cooperatively disrupt axonal transport independently from plaques and tangles. Finally, these results suggest that not all endogenous Aß oligomers trigger the same deleterious changes and that the role of each assembly should be considered separately. SIGNIFICANCE STATEMENT: The mechanistic link between amyloid-ß (Aß) and tau, the two major proteins composing the neuropathological lesions detected in brain tissue of Alzheimer's disease subjects, remains unclear. Here, we report that the trimeric Aß species induce a pathological modification of tau in cultured neurons and in bigenic mice expressing Aß and human tau. This linkage was also observed in postmortem brain tissue from subjects with mild cognitive impairment, when Aß trimers are abundant. Further, this modification of tau was associated with the intracellular accumulation of the precursor protein of Aß, APP, as a result of the selective decrease in kinesin light chain 1 expression. Our findings suggest that Aß trimers might cause axonal transport deficits in AD.

Phosphorylation of FEZ1 by Microtubule Affinity Regulating Kinases regulates its function in presynaptic protein trafficking.

Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer's disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration.

Axonal transport defects are a common phenotype in Drosophila models of ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motor neurons resulting in a catastrophic loss of motor function. Current therapies are severely limited owing to a poor mechanistic understanding of the pathobiology. Mutations in a large number of genes have now been linked to ALS, including SOD1, TARDBP (TDP-43), FUS and C9orf72. Functional analyses of these genes and their pathogenic mutations have provided great insights into the underlying disease mechanisms. Defective axonal transport is hypothesized to be a key factor in the selective vulnerability of motor nerves due to their extraordinary length and evidence that ALS occurs as a distal axonopathy. Axonal transport is seen as an early pathogenic event that precedes cell loss and clinical symptoms and so represents an upstream mechanism for therapeutic targeting. Studies have begun to describe the impact of a few pathogenic mutations on axonal transport but a broad survey across a range of models and cargos is warranted. Here, we assessed the axonal transport of different cargos in multiple Drosophila models of ALS. We found that axonal transport defects are common across all models tested, although they often showed a differential effect between mitochondria and vesicle cargos. Motor deficits were also common across the models and generally worsened with age, though surprisingly there was not a clear correlation between the severity of axonal transport defects and motor ability. These results further support defects in axonal transport as a common factor in models of ALS that may contribute to the pathogenic process.