Microtubule-dependent Transport Research Articles

Poxvirus A51R Proteins Negatively Regulate Microtubule-Dependent Transport by Kinesin-1.

Microtubule (MT)-dependent transport is a critical means of intracellular movement of cellular cargo by kinesin and dynein motors. MT-dependent transport is tightly regulated by cellular MT-associated proteins (MAPs) that directly bind to MTs and either promote or impede motor protein function. Viruses have been widely shown to usurp MT-dependent transport to facilitate their virion movement to sites of replication and/or for exit from the cell. However, it is unclear if viruses also negatively regulate MT-dependent transport. Using single-molecule motility and cellular transport assays, we show that the vaccinia virus (VV)-encoded MAP, A51R, inhibits kinesin-1-dependent transport along MTs in vitro and in cells. This inhibition is selective as the function of kinesin-3 is largely unaffected by VV A51R. Interestingly, we show that A51R promotes the perinuclear accumulation of cellular cargo transported by kinesin-1 such as lysosomes and mitochondria during infection. Moreover, A51R also regulates the release of specialized VV virions that exit the cell using kinesin-1-dependent movement. Using a fluorescently tagged rigor mutant of kinesin-1, we show that these motors accumulate on A51R-stabilized MTs, suggesting these stabilized MTs may form a "kinesin-1 sink" to regulate MT-dependent transport in the cell. Collectively, our findings uncover a new mechanism by which viruses regulate host cytoskeletal processes.

TRIM25 targets p300 for degradation.

p300 is an important transcriptional co-factor. By stimulating the transfer of acetyl residues onto histones and several key transcription factors, p300 enhances transcriptional initiation and impacts cellular processes including cell proliferation and cell division. Despite its importance for cellular homeostasis, its regulation is poorly understood. We show that TRIM25, a member of the TRIM protein family, targets p300 for proteasomal degradation. However, despite TRIM25's RING domain and E3 activity, degradation of p300 by TRIM25 is independent of TRIM25-mediated p300 ubiquitination. Instead, TRIM25 promotes the interaction of p300 with dynein, which ensures a microtubule-dependent transport of p300 to cellular proteasomes. Through mediating p300 degradation, TRIM25 affects p300-dependent gene expression.

Why kiss-and-hop explains that tau does not stabilize microtubules and does not interfere with axonal transport (at physiological conditions).

Tau is a microtubule-associated protein that is enriched in the axonal process of neurons. Post-translational modifications of tau have been implicated in the development of tauopathies characterized by defects in axonal transport, neuronal atrophy, and microtubule disassembly. Although tau is almost quantitatively bound to microtubules under physiological conditions, it does not significantly affect axonal transport. Furthermore, acute or chronic tau deficiency does not result in significant destabilization of neuronal microtubules, challenging the classical view that disease-related tau modifications directly cause axonal microtubule collapse. Here, we discuss how the rapid interaction kinetics of the tau-microtubule interaction, which we previously termed the kiss-and-hop interaction, explains why tau does not affect microtubule-dependent axonal transport but still allows tau to modulate microtubule polymerization. In contrast, tau modifications that slow down the kinetics of the tau-microtubule interaction and increase the residence time of tau at a microtubule interaction site can disrupt axonal transport and cause dendritic atrophy. We discuss the consequences of such a gain-of-toxicity mechanism in terms of the development of disease-modulating drugs that target the tau protein.

Oocytes can repair DNA damage during meiosis viaamicrotubule-dependent recruitment of CIP2A-MDC1-TOPBP1 complex from spindle pole to chromosomes.

Because DNA double-strand breaks (DSBs) greatly threaten genomic integrity, effective DNA damage sensing and repair are essential for cellular survival in all organisms. However, DSB repair mainly occurs during interphase and is repressed during mitosis. Here, we show that, unlike mitotic cells, oocytes can repair DSBs during meiosis I through microtubule-dependent chromosomal recruitment of the CIP2A-MDC1-TOPBP1 complex from spindle poles. After DSB induction, we observed spindle shrinkage and stabilization, as well as BRCA1 and 53BP1 recruitment to chromosomes and subsequent DSB repair during meiosis I. Moreover, p-MDC1 and p-TOPBP1 were recruited from spindle poles to chromosomes in a CIP2A-dependent manner. This pole-to-chromosome relocation of the CIP2A-MDC1-TOPBP1 complex was impaired not only by depolymerizing microtubules but also by depleting CENP-A or HEC1, indicating that the kinetochore/centromere serves as a structural hub for microtubule-dependent transport of the CIP2A-MDC1-TOPBP1 complex. Mechanistically, DSB-induced CIP2A-MDC1-TOPBP1 relocation is regulated by PLK1 but not by ATM activity. Our data provide new insights into the critical crosstalk between chromosomes and spindle microtubules in response to DNA damage to maintain genomic stability during oocyte meiosis.

CYLD deubiquitinates plakoglobin to promote Cx43 membrane targeting and gap junction assembly in the heart

During heart maturation, gap junctions assemble into hemichannels and polarize to the intercalated disc at cell borders to mediate electrical impulse conduction. However, the molecular mechanism underpinning cardiac gap junction assembly remains elusive. Herein, we demonstrate an important role for the deubiquitinating enzyme cylindromatosis (CYLD) in this process. Depletion of CYLD in mice impairs the formation of cardiac gap junctions, accelerates cardiac fibrosis, and increases heart failure. Mechanistically, CYLD interacts with plakoglobin and removes lysine 63-linked polyubiquitin chains from plakoglobin. The deubiquitination ofplakoglobin enhances its interaction with the desmoplakin/end-binding protein 1 complex localized atthe microtubule plus end, thereby promoting microtubule-dependent transport of connexin 43 (Cx43), akey component of gap junctions, to the cell membrane. These findings establish CYLD as a critical playerin regulating gap junction assembly and have important implications in heart development and diseases.

Reduction of kinesin I heavy chain decreases tau hyperphosphorylation, aggregation, and memory impairment in Alzheimer's disease and tauopathy models.

Many neurodegenerative diseases, such as Alzheimer's disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17, are characterized by tau pathology. Numerous motor proteins, many of which are involved in synaptic transmission, mediate transport in neurons. Dysfunction in motor protein-mediated neuronal transport mechanisms occurs in several neurodegenerative disorders but remains understudied in AD. Kinesins are the most important molecular motor proteins required for microtubule-dependent transport in neurons, and kinesin-1 is crucial for neuronal transport among all kinesins. Although kinesin-1 is required for normal neuronal functions, the dysfunction of these motor domains leading to neurodegenerative diseases is not fully understood. Here, we reported that the kinesin-I heavy chain (KIF5B), a key molecular motor protein, is involved in tau homeostasis in AD cells and animal models. We found that the levels of KIF5B in P301S tau mice are high. We also found that the knockdown and knockout (KO) of KIFf5B significantly decreased the tau stability, and overexpression of KIF5B in KIF5B-KO cells significantly increased the expression of phosphorylated and total tau levels. This suggested that KIF5B might prevent tau accumulation. By conducting experiments on P301S tau mice, we showed that partially reducing KIF5B levels can reduce hyperphosphorylation of the human tau protein, formation of insoluble aggregates, and memory impairment. Collectively, our results suggested that decreasing KIF5B levels is sufficient to prevent and/or slow down abnormal tau behavior of AD and other tauopathies.

Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis.

The Fragile-X Mental Retardation Protein (FMRP) is an RNA binding protein that regulates translation of mRNAs essential for synaptic development and plasticity. FMRP interacts with a specific set of mRNAs, aids in their microtubule-dependent transport and regulates their translation through its association with ribosomes. However, the biochemical role of FMRP's domains in forming neuronal granules and associating with microtubules and ribosomes is currently undefined. We report that the C-terminus domain of FMRP is sufficient to bind to ribosomes akin to the full-length protein. Furthermore, the C-terminus domain alone is essential and responsible for FMRP-mediated neuronal translation repression. However, dendritic distribution of FMRP and its microtubule association is favored by the synergistic combination of FMRP domains rather than individual domains. Interestingly, we show that the phosphorylation of hFMRP at Serine-500 is important in modulating the dynamics of translation by controlling ribosome association. This is a fundamental mechanism governing the size and number of FMRP puncta that contain actively translating ribosomes. Finally through the use of pathogenic mutations, we emphasize the hierarchical contribution of FMRP's domains in translation regulation.

Large Rab GTPase Rab44 regulates microtubule-dependent retrograde melanosome transport in melanocytes

Melanosomes are melanin-containing organelles in melanocytes, and they are responsible for skin and hair pigmentation in mammals. The intracellular distribution of melanosomes is mainly determined by the balance between their anterograde transport on actin filaments and retrograde transport on microtubules. Although we have shown previously that melanoregulin and Rab36 serve as cargo receptors on melanosomes for retrograde transport, their knockdown does not completely inhibit retrograde melanosome transport, suggesting the existence of an additional cargo receptor(s) in melanocytes. In this study, we investigated the possible involvement of an atypical large Rab, Rab44, which also contains EF-hand domains and a coiled-coil domain, in retrograde melanosome transport in mouse melanocytes (Rab27A-deficient melan-ash cells). Our results showed that Rab44 localizes on mature melanosomes through lipidation of its C-terminal Rab-like GTPase domain, and that its knockdown results in suppression of retrograde melanosome transport. In addition, our biochemical analysis indicated that Rab44 interacts with the dynein–dynactin motor complex via its coiled-coil domain–containing middle region. Since simultaneous depletion of Rab44, melanoregulin, and Rab36 resulted in almost complete inhibition of retrograde melanosome transport, we propose that Rab44 is the third cargo receptor. We also showed that the N-terminal region of Rab44, which contains EF-hand domains, is required for both retrograde melanosome transport and its Ca2+-modulated activities. Our findings indicated that Rab44 is a third melanosomal cargo receptor, and that, unlike other cargo receptors previously described, its transport function is regulated by Ca2+.

Dynamin-2 regulates microtubule stability via an endocytosis-independent mechanism.

Microtubule stability and dynamics regulations are essential for vital cellular processes, such as microtubule-dependent axonal transport. Dynamin is involved in many membrane fission events, such as clathrin-mediated endocytosis. The ubiquitously expressed dynamin-2 has been reported to regulate microtubule stability. However, the underlying molecular mechanisms remain unclear. This study aimed to investigate the roles of intrinsic properties of dynamin-2 on microtubule regulation by rescue experiments. A heterozygous DNM2 mutation in HeLa cells was generated, and an increase in the level of stabilized microtubules in these heterozygous cells was observed. The expression of wild-type dynamin-2 in heterozygous cells reduced stabilized microtubules. Conversely, the expression of self-assembly-defective mutants of dynamin-2 in the heterozygous cells failed to decrease stabilized microtubules. This indicated that the self-assembling ability of dynamin-2 is necessary for regulating microtubule stability. Moreover, the heterozygous cells expressing the GTPase-defective dynamin-2 mutant, K44A, reduced microtubule stabilization, similar to the cells expressing wild-type dynamin-2, suggesting that GTPase activity of dynamin-2 is not essential for regulating microtubule stability. These results showed that the mechanism of microtubule regulation by dynamin-2 is diverse from that of endocytosis.

Spatial regulation of microtubule-dependent transport by septin GTPases.

The intracellular long-range transport of membrane vesicles and organelles is mediated by microtubule motors (kinesins, dynein) which move cargo with spatiotemporal accuracy and efficiency. How motors navigate the microtubule network and coordinate their activity on membrane cargo are fundamental but poorly understood questions. New studies show that microtubule-dependent membrane traffic is spatially controlled by septins - a unique family of multimerizing GTPases that associate with microtubules and membrane organelles. We review how septins selectively regulate motor interactions with microtubules and membrane cargo. We posit that septins provide a novel traffic code that specifies the movement and directionality of select motor-cargo complexes on distinct microtubule tracks.

Expression and functional analysis of cytoplasmic dynein during spermatogenesis in Portunus trituberculatus

The mechanism of acrosome formation in the crab sperm is a hot topic in crustacean reproduction research. Dynein is a motor protein that performs microtubule-dependent retrograde transport and plays an essential role in spermatogenesis. However, whether cytoplasmic dynein participates in acrosome formation in the crab sperm remains poorly understood. In this study, we cloned the cytoplasmic dynein intermediate chain gene (Pt-DIC) from Portunus trituberculatus testis. Pt-DIC is composed of a p150glued-binding domain, a dynein light chain (DLC)-binding domain, and a dynein heavy chain (DHC)-binding domain. The Pt-DIC gene is widely expressed in different tissues, showing the highest expression in the testis, and it is expressed in different stages of spermatid development, indicating important functions in spermatogenesis. We further observed the colocalization of Pt-DIC and Pt-DHC, Pt-DHC and tubulin, and Pt-DHC and GM130, and the results indicated that cytoplasmic dynein may participate in nuclear shaping and acrosome formation via vesicle transport. In addition, we examined the colocalization of Pt-DHC and a mitochondrion (MT) tracker and that of Pt-DHC and prohibitin (PHB). The results indicated that cytoplasmic dynein participated in mitochondrial transport and mitochondrial degradation. Taken together, these results support the hypothesis that cytoplasmic dynein participates in acrosome formation, nuclear shaping, and mitochondrial transport during spermiogenesis in P. trituberculatus. This study will provide valuable guidance for the artificial fertilization and reproduction of P. trituberculatus.

A kinesin 1-protrudin complex mediates AMPA receptor synaptic removal during long-term depression.

The synaptic removal of AMPA-type glutamate receptors (AMPARs) is a core mechanism for hippocampal long-term depression (LTD). In this study, we address the role of microtubule-dependent transport of AMPARs as a driver for vesicular trafficking and sorting during LTD. Here, we show that the kinesin-1 motor KIF5A/C is strictly required for LTD expression in CA3-to-CA1 hippocampal synapses. Specifically, we find that KIF5 is required for an efficient internalization of AMPARs after NMDA receptor activation. We show that the KIF5/AMPAR complex is assembled in an activity-dependent manner and associates with microsomal membranes upon LTD induction. This interaction is facilitated by the vesicular adaptor protrudin, which is also required for LTD expression. We propose that protrudin links KIF5-dependent transport to endosomal sorting, preventing AMPAR recycling to synapses after LTD induction. Therefore, this work identifies an activity-dependent molecular motor and the vesicular adaptor protein that executes AMPAR synaptic removal during LTD.

Miro1-dependent mitochondrial dynamics in parvalbumin interneurons

The spatiotemporal distribution of mitochondria is crucial for precise ATP provision and calcium buffering required to support neuronal signaling. Fast-spiking GABAergic interneurons expressing parvalbumin (PV+) have a high mitochondrial content reflecting their large energy utilization. The importance for correct trafficking and precise mitochondrial positioning remains poorly elucidated in inhibitory neurons. Miro1 is a Ca²+-sensing adaptor protein that links mitochondria to the trafficking apparatus, for their microtubule-dependent transport along axons and dendrites, in order to meet the metabolic and Ca2+-buffering requirements of the cell. Here, we explore the role of Miro1 in PV+ interneurons and how changes in mitochondrial trafficking could alter network activity in the mouse brain. By employing live and fixed imaging, we found that the impairments in Miro1-directed trafficking in PV+ interneurons altered their mitochondrial distribution and axonal arborization, while PV+ interneuron-mediated inhibition remained intact. These changes were accompanied by an increase in the ex vivo hippocampal γ-oscillation (30-80 Hz) frequency and promoted anxiolysis. Our findings show that precise regulation of mitochondrial dynamics in PV+ interneurons is crucial for proper neuronal signaling and network synchronization.

Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells

Simple SummaryCancer cells release small vesicles called extracellular vesicles (EVs) and these vesicles and their cargo promote cancer progression. The release of these vesicles is coordinated by cellular structures called microtubules. Drugs used in the treatment of breast cancer include eribulin and paclitaxel and they function by inhibiting microtubules. We investigated if these drugs would alter the release of vesicles from breast cancer cells and change their cargo. While our results show that these drugs do not alter the concentration of vesicles released, eribulin but not paclitaxel reduced levels of an important cargo called ILK. This is significant because ILK promotes cancer progression in breast cancer models. These results show that drugs can differently affect the release and cargo of extracellular vesicles and this could be significant for their clinical actions.Extracellular vesicles play a central role in intercellular communication and contribute to cancer progression, including the epithelial-to-mesenchymal transition (EMT). Microtubule targeting agents (MTAs) including eribulin and paclitaxel continue to provide significant value in cancer therapy and their abilities to inhibit oncogenic signaling pathways, including eribulin’s capacity to reverse EMT are being revealed. Because microtubules are involved in the intracellular trafficking required for the formation and cargo loading of small extracellular vesicles (sEVs), we investigated whether MTA-mediated disruption of microtubule-dependent transport would impact sEV release and their cargo. Eribulin and paclitaxel caused an intracellular accumulation of CD63, a tetraspanin component of sEVs, in late/multivesicular endosomes of triple-negative breast cancer cells, consistent with the disruption of endosomal sorting and exosome cargo loading in these cells. While the concentrations of sEVs released from MTA-treated cells were not significantly altered, levels of CD63 and the CD63-associated cargos, ILK and β-integrin, were reduced in sEVs isolated from eribulin-treated HCC1937 cells as compared to vehicle or paclitaxel-treated cells. These results show that eribulin can reduce specific sEV cargos, including ILK, a major transducer of EMT in the tumor microenvironment, which may contribute to eribulin’s ability to reverse EMT to promote anticancer efficacy.

Folylpoly-ɣ-glutamate synthetase association to the cytoskeleton: Implications to folate metabolon compartmentalization

Folates are essential for nucleotide biosynthesis, amino acid metabolism and cellular proliferation. Following carrier-mediated uptake, folates are polyglutamylated by folylpoly-ɣ-glutamate synthetase (FPGS), resulting in their intracellular retention. FPGS appears as a long isoform, directed to mitochondria via a leader sequence, and a short isoform reported as a soluble cytosolic protein (cFPGS). However, since folates are labile and folate metabolism is compartmentalized, we herein hypothesized that cFPGS is associated with the cytoskeleton, to couple folate uptake and polyglutamylation and channel folate polyglutamates to metabolon compartments. We show that cFPGS is a cytoskeleton-microtubule associated protein: Western blot analysis revealed that endogenous cFPGS is associated with the insoluble cellular fraction, i.e., cytoskeleton and membranes, but not with the cytosol. Mass spectrometry analysis identified the putative cFPGS interactome primarily consisting of microtubule subunits and cytoskeletal motor proteins. Consistently, immunofluorescence microscopy with cytosol-depleted cells demonstrated the association of cFPGS with the cytoskeleton and unconventional myosin-1c. Furthermore, since anti-microtubule, anti-actin cytoskeleton, and coatomer dissociation-inducing agents yielded perinuclear pausing of cFPGS, we propose an actin- and microtubule-dependent transport of cFPGS between the ER-Golgi and the plasma membrane. These novel findings support the coupling of folate transport with polyglutamylation and folate channeling to intracellular metabolon compartments. SignificanceFPGS, an essential enzyme catalyzing intracellular folate polyglutamylation and efficient retention, was described as a soluble cytosolic enzyme in the past 40 years. However, based on the lability of folates and the compartmentalization of folate metabolism and nucleotide biosynthesis, we herein hypothesized that cytoplasmic FPGS is associated with the cytoskeleton, to couple folate transport and polyglutamylation as well as channel folate polyglutamates to biosynthetic metabolon compartments. Indeed, using complementary techniques including Mass-spectrometry proteomics and fluorescence microscopy, we show that cytoplasmic FPGS is associated with the cytoskeleton and unconventional myosin-1c. This novel cytoskeletal localization of cytoplasmic FPGS supports the dynamic channeling of polyglutamylated folates to metabolon compartments to avoid oxidation and intracellular dilution of folates, while enhancing folate-dependent de novo biosynthesis of nucleotides and DNA/protein methylation.

The Deubiquitinating Enzyme Ataxin-3 Regulates Ciliogenesis and Phagocytosis in the Retina

SUMMARYExpansion of a CAG repeat in ATXN3 causes the dominant polyglutamine disease spinocerebellar ataxia type 3 (SCA3), yet the physiological role of ATXN3 remains unclear. Here, we focus on unveiling the function of Ataxin-3 (ATXN3) in the retina, a neurological organ amenable to morphological and physiological studies. Depletion of Atxn3 in zebrafish and mice causes morphological and functional retinal alterations and, more precisely, photoreceptor cilium and outer segment elongation, cone opsin mislocalization, and cone hyperexcitation. ATXN3 localizes at the basal body and axoneme of the cilium, supporting its role in regulating ciliary length. Abrogation of Atxn3 expression causes decreased levels of the regulatory protein KEAP1 in the retina and delayed phagosome maturation in the retinal pigment epithelium. We propose that ATXN3 regulates two relevant biological processes in the retina, namely, ciliogenesis and phagocytosis, by modulating microtubule polymerization and microtubule-dependent retrograde transport, thus positing ATXN3 as a causative or modifier gene in retinal/macular dystrophies.

Aptamer-Functionalized Gold Nanostars for on-Demand Delivery of Anticancer Therapeutics.

Gold nanostars (AuNS) are promising carriers for targeted delivery of therapeutic oligonucleotides, but their potential in fabricating an on-demand drug release system in a facile and robust way remains to be explored. In this paper, we used a model aptamer (HApt), acting not only as a target ligand but also as a natural thermal-responsive material, to decorate AuNS. The prepared gold nanoconstruct, HApt@AuNS, displayed stoichiometric loading capacity of the anthracycline drug doxorubicin (Dox). The on-demand drug release was realized by illuminating nanoconstructs with near-infrared (NIR) light. Furthermore, a higher degree of Dox release from the nanoconstructs was achieved in an acidic environment, compared to neutral conditions. The in vitro experiments showed that Dox-intercalation did not affect the cell uptake efficiency of HApt@AuNS, which could enter cells through clathrin-mediated endocytosis and microtubule-dependent active transport to lysosomes. Dox-loaded HApt@AuNS exhibited intracellular on-demand drug release and enhanced toxicity against cancer cells by NIR-irradiation.

Deletion of the Pseudorabies Virus gE/gI-US9p complex disrupts kinesin KIF1A and KIF5C recruitment during egress, and alters the properties of microtubule-dependent transport in vitro.

During infection of neurons by alphaherpesviruses including Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) viral nucleocapsids assemble in the cell nucleus, become enveloped in the cell body then traffic into and down axons to nerve termini for spread to adjacent epithelia. The viral membrane protein US9p and the membrane glycoprotein heterodimer gE/gI play critical roles in anterograde spread of both HSV-1 and PRV, and several models exist to explain their function. Biochemical studies suggest that PRV US9p associates with the kinesin-3 motor KIF1A in a gE/gI-stimulated manner, and the gE/gI-US9p complex has been proposed to recruit KIF1A to PRV for microtubule-mediated anterograde trafficking into or along the axon. However, as loss of gE/gI-US9p essentially abolishes delivery of alphaherpesviruses to the axon it is difficult to determine the microtubule-dependent trafficking properties and motor-composition of Δ(gE/gI−US9p) particles. Alternatively, studies in HSV-1 have suggested that gE/gI and US9p are required for the appearance of virions in the axon because they act upstream, to help assemble enveloped virions in the cell body. We prepared Δ(gE/gI-US9p) mutant, and control parental PRV particles from differentiated cultured neuronal or porcine kidney epithelial cells and quantitated the efficiency of virion assembly, the properties of microtubule-dependent transport and the ability of viral particles to recruit kinesin motors. We find that loss of gE/gI-US9p has no significant effect upon PRV particle assembly but leads to greatly diminished plus end-directed traffic, and enhanced minus end-directed and bidirectional movement along microtubules. PRV particles prepared from infected differentiated mouse CAD neurons were found to be associated with either kinesin KIF1A or kinesin KIF5C, but not both. Loss of gE/gI-US9p resulted in failure to recruit KIF1A and KF5C, but did not affect dynein binding. Unexpectedly, while KIF5C was expressed in undifferentiated and differentiated CAD neurons it was only found associated with PRV particles prepared from differentiated cells.

Oxidative Stress-Induced Axon Fragmentation Is a Consequence of Reduced Axonal Transport in Hereditary Spastic Paraplegia SPAST Patient Neurons

Hereditary spastic paraplegia (HSP) is a group of inherited disorders characterized by progressive spasticity and paralysis of the lower limbs. Autosomal dominant mutations in SPAST gene account for ∼40% of adult-onset patients. We have previously shown that SPAST patient cells have reduced organelle transport and are therefore more sensitive to oxidative stress. To test whether these effects are present in neuronal cells, we first generated 11 induced pluripotent stem (iPS) cell lines from fibroblasts of three healthy controls and three HSP patients with different SPAST mutations. These cells were differentiated into FOXG1-positive forebrain neurons and then evaluated for multiple aspects of axonal transport and fragmentation. Patient neurons exhibited reduced levels of SPAST encoded spastin, as well as a range of axonal deficits, including reduced levels of stabilized microtubules, lower peroxisome transport speed as a consequence of reduced microtubule-dependent transport, reduced number of peroxisomes, and higher density of axon swellings. Patient axons fragmented significantly more than controls following hydrogen peroxide exposure, suggesting for the first time that the SPAST patient axons are more sensitive than controls to the deleterious effects of oxidative stress. Treatment of patient neurons with tubulin-binding drugs epothilone D and noscapine rescued axon peroxisome transport and protected them against axon fragmentation induced by oxidative stress, showing that SPAST patient axons are vulnerable to oxidative stress-induced degeneration as a consequence of reduced axonal transport.

Loss of BICD2 in muscle drives motor neuron loss in a developmental form of spinal muscular atrophy

Autosomal dominant missense mutations in BICD2 cause Spinal Muscular Atrophy Lower Extremity Predominant 2 (SMALED2), a developmental disease of motor neurons. BICD2 is a key component of the cytoplasmic dynein/dynactin motor complex, which in axons drives the microtubule-dependent retrograde transport of intracellular cargo towards the cell soma. Patients with pathological mutations in BICD2 develop malformations of cortical and cerebellar development similar to Bicd2 knockout (−/−) mice. In this study we sought to re-examine the motor neuron phenotype of conditional Bicd2−/− mice. Bicd2−/− mice show a significant reduction in the number of large calibre motor neurons of the L4 ventral root compared to wild type mice. Muscle-specific knockout of Bicd2 results in a similar reduction in L4 ventral axons comparable to global Bicd2−/− mice. Rab6, a small GTPase required for the sorting of exocytic vesicles from the Trans Golgi Network to the plasma membrane is a major binding partner of BICD2. We therefore examined the secretory pathway in SMALED2 patient fibroblasts and demonstrated that BICD2 is required for physiological flow of constitutive secretory cargoes from the Trans Golgi Network to the plasma membrane using a VSV-G reporter assay. Together, these data indicate that BICD2 loss from muscles is a major driver of non-cell autonomous pathology in the motor nervous system, which has important implications for future therapeutic approaches in SMALED2.