Publications by authors named "Giampietro Schiavo"

133 Publications

Walking the line: mechanisms underlying directional mRNA transport and localisation in neurons and beyond.

Cell Mol Life Sci 2020 Dec 20. Epub 2020 Dec 20.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK.

Messenger RNA (mRNA) localisation enables a high degree of spatiotemporal control on protein synthesis, which contributes to establishing the asymmetric protein distribution required to set up and maintain cellular polarity. As such, a tight control of mRNA localisation is essential for many biological processes during development and in adulthood, such as body axes determination in Drosophila melanogaster and synaptic plasticity in neurons. The mechanisms controlling how mRNAs are localised, including diffusion and entrapment, local degradation and directed active transport, are largely conserved across evolution and have been under investigation for decades in different biological models. In this review, we will discuss the standing of the field regarding directional mRNA transport in light of the recent discovery that RNA can hitchhike on cytoplasmic organelles, such as endolysosomes, and the impact of these transport modalities on our understanding of neuronal function during development, adulthood and in neurodegeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-020-03724-3DOI Listing
December 2020

Potential human transmission of amyloid β pathology: surveillance and risks.

Lancet Neurol 2020 10 16;19(10):872-878. Epub 2020 Sep 16.

VIB-KU Leuven Center for Brain and Disease Research, KU Leuven, Leuven, Belgium; Department of Neurosciences, Leuven Brain Institute, KU Leuven, Leuven, Belgium; UK Dementia Research Institute, University College London, London, UK. Electronic address:

Studies in experimental animals show transmissibility of amyloidogenic proteins associated with prion diseases, Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases. Although these data raise potential concerns for public health, convincing evidence for human iatrogenic transmission only exists for prions and amyloid β after systemic injections of contaminated growth hormone extracts or dura mater grafts derived from cadavers. Even though these procedures are now obsolete, some reports raise the possibility of iatrogenic transmission of amyloid β through putatively contaminated neurosurgical equipment. Iatrogenic transmission of amyloid β might lead to amyloid deposition in the brain parenchyma and blood vessel walls, potentially resulting in cerebral amyloid angiopathy after several decades. Cerebral amyloid angiopathy can cause life-threatening brain haemorrhages; yet, there is no proof that the transmission of amyloid β can also lead to Alzheimer's dementia. Large, long-term epidemiological studies and sensitive, cost-efficient tools to detect amyloid are needed to better understand any potential routes of amyloid β transmission and to clarify whether other similar proteopathic seeds, such as tau or α-synuclein, can also be transferred iatrogenically.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S1474-4422(20)30238-6DOI Listing
October 2020

Altered Sensory Neuron Development in CMT2D Mice Is Site-Specific and Linked to Increased GlyRS Levels.

Front Cell Neurosci 2020 11;14:232. Epub 2020 Aug 11.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, United Kingdom.

Dominant, missense mutations in the widely and constitutively expressed gene cause peripheral neuropathy that usually begins in adolescence and principally impacts the upper limbs. Caused by a toxic gain-of-function in the encoded glycyl-tRNA synthetase (GlyRS) enzyme, the neuropathology appears to be independent of the canonical role of GlyRS in aminoacylation. Patients display progressive, life-long weakness and wasting of muscles in hands followed by feet, with frequently associated deficits in sensation. When dysfunction is observed in motor and sensory nerves, there is a diagnosis of Charcot-Marie-Tooth disease type 2D (CMT2D), or distal hereditary motor neuropathy type V if the symptoms are purely motor. The cause of this varied sensory involvement remains unresolved, as are the pathomechanisms underlying the selective neurodegeneration characteristic of the disease. We have previously identified in CMT2D mice that neuropathy-causing mutations perturb sensory neuron fate and permit mutant GlyRS to aberrantly interact with neurotrophin receptors (Trks). Here, we extend this work by interrogating further the anatomy and function of the CMT2D sensory nervous system in mutant mice, obtaining several key results: (1) sensory pathology is restricted to neurons innervating the hindlimbs; (2) perturbation of sensory development is not common to all mouse models of neuromuscular disease; (3) axonal transport of signaling endosomes is not impaired in afferent neurons of all CMT2D mouse models; and (4) expression is selectively elevated in a subset of sensory neurons and linked to sensory developmental defects. These findings highlight the importance of comparative neurological assessment in mouse models of disease and shed light on key proposed neuropathogenic mechanisms in -linked neuropathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2020.00232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431706PMC
August 2020

Knockin' on heaven's door: Molecular mechanisms of neuronal tau uptake.

J Neurochem 2021 Mar 1;156(5):563-588. Epub 2020 Sep 1.

UK Dementia Research Institute, University College London, London, UK.

Since aggregates of the microtubule-binding protein tau were found to be the main component of neurofibrillary tangles more than 30 years ago, their contribution to neurodegeneration in Alzheimer's disease (AD) and tauopathies has become well established. Recent work shows that both tau load and its distribution in the brain of AD patients correlate with cognitive decline more closely compared to amyloid plaque deposition. In addition, the amyloid cascade hypothesis has been recently challenged because of disappointing results of clinical trials designed to treat AD by reducing beta-amyloid levels, thus fuelling a renewed interest in tau. There is now robust evidence to indicate that tau pathology can spread within the central nervous system via a prion-like mechanism following a stereotypical pattern, which can be explained by the trans-synaptic inter-neuronal transfer of pathological tau. In the receiving neuron, tau has been shown to take multiple routes of internalisation, which are partially dependent on its conformation and aggregation status. Here, we review the emerging mechanisms proposed for the uptake of extracellular tau in neurons and the requirements for the propagation of its pathological conformers, addressing how they gain access to physiological tau monomers in the cytosol. Furthermore, we highlight some of the key mechanistic gaps of the field, which urgently need to be addressed to expand our understanding of tau propagation and lead to the identification of new therapeutic strategies for tauopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jnc.15144DOI Listing
March 2021

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons.

J Vis Exp 2020 07 11(161). Epub 2020 Jul 11.

Department of Physiology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile; Institute of Biomedical Sciences. Faculty of Medicine and Faculty of Life Sciences, Universidad Andrés Bello;

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity. Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/61262DOI Listing
July 2020

Developmental demands contribute to early neuromuscular degeneration in CMT2D mice.

Cell Death Dis 2020 07 23;11(7):564. Epub 2020 Jul 23.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK.

Dominantly inherited, missense mutations in the widely expressed housekeeping gene, GARS1, cause Charcot-Marie-Tooth type 2D (CMT2D), a peripheral neuropathy characterised by muscle weakness and wasting in limb extremities. Mice modelling CMT2D display early and selective neuromuscular junction (NMJ) pathology, epitomised by disturbed maturation and neurotransmission, leading to denervation. Indeed, the NMJ disruption has been reported in several different muscles; however, a systematic comparison of neuromuscular synapses from distinct body locations has yet to be performed. We therefore analysed NMJ development and degeneration across five different wholemount muscles to identify key synaptic features contributing to the distinct pattern of neurodegeneration in CMT2D mice. Denervation was found to occur along a distal-to-proximal gradient, providing a cellular explanation for the greater weakness observed in mutant Gars hindlimbs compared with forelimbs. Nonetheless, muscles from similar locations and innervated by axons of equivalent length showed significant differences in neuropathology, suggestive of additional factors impacting on site-specific neuromuscular degeneration. Defective NMJ development preceded and associated with degeneration, but was not linked to a delay of wild-type NMJ maturation processes. Correlation analyses indicate that muscle fibre type nor synaptic architecture explain the differential denervation of CMT2D NMJs, rather it is the extent of post-natal synaptic growth that predisposes to neurodegeneration. Together, this work improves our understanding of the mechanisms driving synaptic vulnerability in CMT2D and hints at pertinent pathogenic pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-020-02798-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378196PMC
July 2020

A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels.

BMC Res Notes 2020 Jun 24;13(1):302. Epub 2020 Jun 24.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK.

Objective: Dorsal root ganglia (DRG) are heterogeneous assemblies of assorted sensory neuron cell bodies found in bilateral pairs at every level of the spinal column. Pseudounipolar afferent neurons convert external stimuli from the environment into electrical signals that are retrogradely transmitted to the spinal cord dorsal horn. To do this, they extend single axons from their DRG-resident somas that then bifurcate and project both centrally and distally. DRG can be dissected from mice at embryonic stages and any age post-natally, and have been extensively used to study sensory neuron development and function, response to injury, and pathological processes in acquired and genetic diseases. We have previously published a step-by-step dissection method for the rapid isolation of post-natal mouse DRG. Here, the objective is to extend the protocol by providing training videos that showcase the dissection in fine detail and permit the extraction of ganglia from defined spinal levels.

Results: By following this method, the reader will be able to swiftly and accurately isolate specific lumbar, thoracic, and cervical DRG from mice. Dissected ganglia can then be used for RNA/protein analyses, subjected to immunohistochemical examination, and cultured as explants or dissociated primary neurons, for in-depth investigations of sensory neuron biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13104-020-05147-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313212PMC
June 2020

Morphological variability is greater at developing than mature mouse neuromuscular junctions.

J Anat 2020 Oct 13;237(4):603-617. Epub 2020 Jun 13.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, UK.

The neuromuscular junction (NMJ) is the highly specialised peripheral synapse formed between lower motor neuron terminals and muscle fibres. Post-synaptic acetylcholine receptors (AChRs), which are found in high density in the muscle membrane, bind to acetylcholine released into the synaptic cleft of the NMJ, thereby enabling the conversion of motor action potentials to muscle contractions. NMJs have been studied for many years as a general model for synapse formation, development and function, and are known to be early sites of pathological changes in many neuromuscular diseases. However, information is limited on the diversity of NMJs in different muscles, how synaptic morphology changes during development, and the relevance of these parameters to neuropathology. Here, this crucial gap was addressed using a robust and standardised semi-automated workflow called NMJ-morph to quantify features of pre- and post-synaptic NMJ architecture in an unbiased manner. Five wholemount muscles from wild-type mice were dissected and compared at immature (post-natal day, P7) and early adult (P31-32) timepoints. The inter-muscular variability was greater in mature post-synaptic AChR morphology than that of the pre-synaptic motor neuron terminal. Moreover, the developing NMJ showed greater differences across muscles than the mature synapse, perhaps due to the observed distinctions in synaptic growth between muscles. Nevertheless, the amount of nerve to muscle contact was consistent, suggesting that pathological denervation can be reliably compared across different muscles in mouse models of neurodegeneration. Additionally, mature post-synaptic endplate diameters correlated with fibre type, independently of muscle fibre diameter. Altogether, this work provides detailed information on healthy pre- and post-synaptic NMJ morphology from five anatomically and functionally distinct mouse muscles, delivering useful reference data for future comparison with neuromuscular disease models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/joa.13228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495279PMC
October 2020

In Vivo Imaging of Anterograde and Retrograde Axonal Transport in Rodent Peripheral Nerves.

Methods Mol Biol 2020 ;2143:271-292

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, UK.

Axonal transport, which is the process mediating the active shuttling of a variety cargoes from one end of an axon to the other, is essential for the development, function, and survival of neurons. Impairments in this dynamic process are linked to diverse nervous system diseases and advanced ageing. It is thus essential that we quantitatively study the kinetics of axonal transport to gain an improved understanding of neuropathology as well as the molecular and cellular mechanisms regulating cargo trafficking. One of the best ways to achieve this goal is by imaging individual, fluorescent cargoes in live systems and analyzing the kinetic properties of their progression along the axon. We have therefore developed an intravital technique to visualize different organelles, such as signaling endosomes and mitochondria, being actively transported in the axons of both motor and sensory neurons in live, anesthetized rodents. In this chapter, we provide step-by-step instructions on how to deliver specific organelle-targeting, fluorescent probes using several routes of administration to image individual cargoes being bidirectionally transported along axons within the exposed sciatic nerve. This method can provide detailed, physiologically relevant information on axonal transport, and is thus poised to elucidate mechanisms regulating this process in both health and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-0716-0585-1_20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116510PMC
January 2020

FUS ALS-causative mutations impair FUS autoregulation and splicing factor networks through intron retention.

Nucleic Acids Res 2020 07;48(12):6889-6905

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK.

Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337901PMC
July 2020

Mice Carrying ALS Mutant TDP-43, but Not Mutant FUS, Display In Vivo Defects in Axonal Transport of Signaling Endosomes.

Cell Rep 2020 03;30(11):3655-3662.e2

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK; UK Dementia Research Institute, University College London, London WC1E 6BT, UK; Discoveries Centre for Regenerative and Precision Medicine, University College London Campus, London WC1N 3BG, UK. Electronic address:

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in axonal transport have been identified in several ALS models, but in vivo evidence remains limited, thus their pathogenetic importance remains to be fully resolved. We therefore analyzed the in vivo dynamics of retrogradely transported, neurotrophin-containing signaling endosomes in nerve axons of two ALS mouse models with mutations in the RNA processing genes TARDBP and FUS. TDP-43 mice, which show neuromuscular pathology without motor neuron loss, display axonal transport perturbations manifesting between 1.5 and 3 months and preceding symptom onset. Contrastingly, despite 20% motor neuron loss, transport remained largely unaffected in Fus mice. Deficiencies in retrograde axonal transport of signaling endosomes are therefore not shared by all ALS-linked genes, indicating that there are mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.02.078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090381PMC
March 2020

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

Acta Neuropathol Commun 2020 03 17;8(1):34. Epub 2020 Mar 17.

UCL Queen Square Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, UK.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40478-020-00909-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076953PMC
March 2020

Duplication of clostridial binding domains for enhanced macromolecular delivery into neurons.

Toxicon X 2020 Mar;5:100019

Department of Biomedical Science, University of Sheffield, Sheffield, S10 2TN, UK.

Neurological diseases constitute a quarter of global disease burden and are expected to rise worldwide with the ageing of human populations. There is an increasing need to develop new molecular systems which can deliver drugs specifically into neurons, non-dividing cells meant to last a human lifetime. Neuronal drug delivery must rely on agents which can recognise neurons with high specificity and affinity. Here we used a recently introduced 'stapling' system to prepare macromolecules carrying duplicated binding domains from the clostridial family of neurotoxins. We engineered individual parts of clostridial neurotoxins separately and combined them using a strong alpha-helical bundle. We show that combining two identical binding domains of tetanus and botulinum type D neurotoxins, in a sterically defined way by protein stapling, allows enhanced intracellular delivery of molecules into neurons. We also engineered a botulinum neurotoxin type C variant with a duplicated binding domain which increased enzymatic delivery compared to the native type C toxin. We conclude that duplication of the binding parts of tetanus or botulinum neurotoxins will allow production of high avidity agents which could deliver imaging reagents and large therapeutic enzymes into neurons with superior efficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxcx.2019.100019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7043326PMC
March 2020

DYNLRB1 is essential for dynein mediated transport and neuronal survival.

Neurobiol Dis 2020 07 20;140:104816. Epub 2020 Feb 20.

Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel.

The cytoplasmic dynein motor complex transports essential signals and organelles from the cell periphery to the perinuclear region, hence is critical for the survival and function of highly polarized cells such as neurons. Dynein Light Chain Roadblock-Type 1 (DYNLRB1) is thought to be an accessory subunit required for specific cargos, but here we show that it is essential for general dynein-mediated transport and sensory neuron survival. Homozygous Dynlrb1 null mice are not viable and die during early embryonic development. Furthermore, heterozygous or adult knockdown animals display reduced neuronal growth, and selective depletion of Dynlrb1 in proprioceptive neurons compromises their survival. Conditional depletion of Dynlrb1 in sensory neurons causes deficits in several signaling pathways, including β-catenin subcellular localization, and a severe impairment in the axonal transport of both lysosomes and retrograde signaling endosomes. Hence, DYNLRB1 is an essential component of the dynein complex, and given dynein's critical functions in neuronal physiology, DYNLRB1 could have a prominent role in the etiology of human neurodegenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nbd.2020.104816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273200PMC
July 2020

PTPN23 binds the dynein adaptor BICD1 and is required for endocytic sorting of neurotrophin receptors.

J Cell Sci 2020 03 30;133(6). Epub 2020 Mar 30.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK

Signalling by target-derived neurotrophins is essential for the correct development of the nervous system and its maintenance throughout life. Several aspects concerning the lifecycle of neurotrophins and their receptors have been characterised over the years, including the formation, endocytosis and trafficking of signalling-competent ligand-receptor complexes. However, the molecular mechanisms directing the sorting of activated neurotrophin receptors are still elusive. Previously, our laboratory identified Bicaudal-D1 (BICD1), a dynein motor adaptor, as a key factor for lysosomal degradation of brain-derived neurotrophic factor (BDNF)-activated TrkB (also known as NTRK2) and p75 (also known as NGFR) in motor neurons. Here, using a proteomics approach, we identified protein tyrosine phosphatase, non-receptor type 23 (PTPN23), a member of the endosomal sorting complexes required for transport (ESCRT) machinery, in the BICD1 interactome. Molecular mapping revealed that PTPN23 is not a canonical BICD1 cargo; instead, PTPN23 binds the N-terminus of BICD1, which is also essential for the recruitment of cytoplasmic dynein. In line with the BICD1-knockdown phenotype, loss of PTPN23 leads to increased accumulation of BDNF-activated p75 and TrkB in swollen vacuole-like compartments, suggesting that neuronal PTPN23 is a novel regulator of the endocytic sorting of neurotrophin receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.242412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132798PMC
March 2020

IGF1R regulates retrograde axonal transport of signalling endosomes in motor neurons.

EMBO Rep 2020 03 7;21(3):e49129. Epub 2020 Feb 7.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London, UK.

Signalling endosomes are essential for trafficking of activated ligand-receptor complexes and their distal signalling, ultimately leading to neuronal survival. Although deficits in signalling endosome transport have been linked to neurodegeneration, our understanding of the mechanisms controlling this process remains incomplete. Here, we describe a new modulator of signalling endosome trafficking, the insulin-like growth factor 1 receptor (IGF1R). We show that IGF1R inhibition increases the velocity of signalling endosomes in motor neuron axons, both in vitro and in vivo. This effect is specific, since IGF1R inhibition does not alter the axonal transport of mitochondria or lysosomes. Our results suggest that this change in trafficking is linked to the dynein adaptor bicaudal D1 (BICD1), as IGF1R inhibition results in an increase in the de novo synthesis of BICD1 in the axon of motor neurons. Finally, we found that IGF1R inhibition can improve the deficits in signalling endosome transport observed in a mouse model of amyotrophic lateral sclerosis (ALS). Taken together, these findings suggest that IGF1R inhibition may be a new therapeutic target for ALS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.201949129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054680PMC
March 2020

Travelling Together: A Unifying Pathomechanism for ALS.

Trends Neurosci 2020 01 16;43(1):1-2. Epub 2019 Nov 16.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK; UK Dementia Research Institute, University College London, London WC1E 6BT, UK; Discoveries Centre for Regenerative and Precision Medicine, University College London Campus, London WC1N 3BG, UK. Electronic address:

Axonal transport is critical for neuronal homeostasis and relies on motor complexes bound to cargoes via specific adaptors. However, the mechanisms responsible for the spatiotemporal regulation of axonal transport are not completely understood. A recent study by Liao et al. contributes to filling this gap by reporting that RNA granules 'hitchhike' on LAMP1-positive organelles using annexin A11 as a tether.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tins.2019.10.006DOI Listing
January 2020

The evolution of the axonal transport toolkit.

Traffic 2020 01 28;21(1):13-33. Epub 2019 Nov 28.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, UK.

Neurons are highly polarized cells that critically depend on long-range, bidirectional transport between the cell body and synapse for their function. This continual and highly coordinated trafficking process, which takes place via the axon, has fascinated researchers since the early 20th century. Ramon y Cajal first proposed the existence of axonal trafficking of biological material after observing that dissociation of the axon from the cell body led to neuronal degeneration. Since these first indirect observations, the field has come a long way in its understanding of this fundamental process. However, these advances in our knowledge have been aided by breakthroughs in other scientific disciplines, as well as the parallel development of novel tools, techniques and model systems. In this review, we summarize the evolution of tools used to study axonal transport and discuss how their deployment has refined our understanding of this process. We also highlight innovative tools currently being developed and how their addition to the available axonal transport toolkit might help to address key outstanding questions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tra.12710DOI Listing
January 2020

TSPAN5 Enriched Microdomains Provide a Platform for Dendritic Spine Maturation through Neuroligin-1 Clustering.

Cell Rep 2019 10;29(5):1130-1146.e8

CNR, Institute of Neuroscience, Milan 20129, Italy. Electronic address:

Tetraspanins are a class of evolutionarily conserved transmembrane proteins with 33 members identified in mammals that have the ability to organize specific membrane domains, named tetraspanin-enriched microdomains (TEMs). Despite the relative abundance of different tetraspanins in the CNS, few studies have explored their role at synapses. Here, we investigate the function of TSPAN5, a member of the tetraspanin superfamily for which mRNA transcripts are found at high levels in the mouse brain. We demonstrate that TSPAN5 is localized in dendritic spines of pyramidal excitatory neurons and that TSPAN5 knockdown induces a dramatic decrease in spine number because of defects in the spine maturation process. Moreover, we show that TSPAN5 interacts with the postsynaptic adhesion molecule neuroligin-1, promoting its correct surface clustering. We propose that membrane compartmentalization by tetraspanins represents an additional mechanism for regulating excitatory synapses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.09.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899445PMC
October 2019

Axonal transport and neurological disease.

Nat Rev Neurol 2019 12 26;15(12):691-703. Epub 2019 Sep 26.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, UK.

Axonal transport is the process whereby motor proteins actively navigate microtubules to deliver diverse cargoes, such as organelles, from one end of the axon to the other, and is widely regarded as essential for nerve development, function and survival. Mutations in genes encoding key components of the transport machinery, including motor proteins, motor adaptors and microtubules, have been discovered to cause neurological disease. Moreover, disruptions in axonal cargo trafficking have been extensively reported across a wide range of nervous system disorders. However, whether these impairments have a major causative role in, are contributing to or are simply a consequence of neuronal degeneration remains unclear. Therefore, the fundamental relevance of defective trafficking along axons to nerve dysfunction and pathology is often debated. In this article, we review the latest evidence emerging from human and in vivo studies on whether perturbations in axonal transport are indeed integral to the pathogenesis of neurological disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41582-019-0257-2DOI Listing
December 2019

Differential regulation of Kidins220 isoforms in Huntington's disease.

Brain Pathol 2020 01 16;30(1):120-136. Epub 2019 Jul 16.

Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by brain atrophy particularly in the striatum that produces motor impairment, and cognitive and psychiatric disturbances. Multiple pathogenic mechanisms have been proposed including dysfunctions in neurotrophic support and calpain-overactivation, among others. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is an essential mediator of neurotrophin signaling. In adult brain, Kidins220 presents two main isoforms that differ in their carboxy-terminal length and critical protein-protein interaction domains. These variants are generated through alternative terminal exon splicing of the conventional exon 32 (Kidins220-C32) and the recently identified exon 33 (Kidins220-C33). The lack of domains encoded by exon 32 involved in key neuronal functions, including those controlling neurotrophin pathways, pointed to Kidins220-C33 as a form detrimental for neurons. However, the functional role of Kidins220-C33 in neurodegeneration or other pathologies, including HD, has not been explored. In the present work, we discover an unexpected selective downregulation of Kidins220-C33, in the striatum of HD patients, as well as in the R6/1 HD mouse model starting at early symptomatic stages. These changes are C33-specific as Kidins220-C32 variant remains unchanged. We also find the early decrease in Kidins220-C33 levels takes place in neurons, suggesting an unanticipated neuroprotective role for this isoform. Finally, using ex vivo assays and primary neurons, we demonstrate that Kidins220-C33 is downregulated by mechanisms that depend on the activation of the protease calpain. Altogether, these results strongly suggest that calpain-mediated Kidins220-C33 proteolysis modulates onset and/or progression of HD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bpa.12761DOI Listing
January 2020

Deacetylation of Miro1 by HDAC6 blocks mitochondrial transport and mediates axon growth inhibition.

J Cell Biol 2019 06 8;218(6):1871-1890. Epub 2019 May 8.

Department of Biological Sciences, University of South Carolina, Columbia, SC

Inhibition of histone deacetylase 6 (HDAC6) was shown to support axon growth on the nonpermissive substrates myelin-associated glycoprotein (MAG) and chondroitin sulfate proteoglycans (CSPGs). Though HDAC6 deacetylates α-tubulin, we find that another HDAC6 substrate contributes to this axon growth failure. HDAC6 is known to impact transport of mitochondria, and we show that mitochondria accumulate in distal axons after HDAC6 inhibition. Miro and Milton proteins link mitochondria to motor proteins for axon transport. Exposing neurons to MAG and CSPGs decreases acetylation of Miro1 on Lysine 105 (K105) and decreases axonal mitochondrial transport. HDAC6 inhibition increases acetylated Miro1 in axons, and acetyl-mimetic Miro1 K105Q prevents CSPG-dependent decreases in mitochondrial transport and axon growth. MAG- and CSPG-dependent deacetylation of Miro1 requires RhoA/ROCK activation and downstream intracellular Ca increase, and Miro1 K105Q prevents the decrease in axonal mitochondria seen with activated RhoA and elevated Ca These data point to HDAC6-dependent deacetylation of Miro1 as a mediator of axon growth inhibition through decreased mitochondrial transport.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201702187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548128PMC
June 2019

Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy.

Mol Cell 2019 04 7;74(2):347-362.e6. Epub 2019 Mar 7.

Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2019.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642318PMC
April 2019

Toxins 2017 conference in Madrid.

Toxicon 2018 Jun 20;147. Epub 2018 Mar 20.

University College London, Sobell Department of Motor Neuroscience and Movement Disorders, London, United Kingdom.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxicon.2018.03.010DOI Listing
June 2018

Retrograde transport of Akt by a neuronal Rab5-APPL1 endosome.

Sci Rep 2019 02 21;9(1):2433. Epub 2019 Feb 21.

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307, Dresden, Germany.

Long-distance axonal trafficking plays a critical role in neuronal function and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-38637-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385319PMC
February 2019

UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy.

Brain 2018 10;141(10):2878-2894

Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, UK.

Deafferentation of motor neurons as a result of defective sensory-motor connectivity is a critical early event in the pathogenesis of spinal muscular atrophy, but the underlying molecular pathways remain unknown. We show that restoration of ubiquitin-like modifier-activating enzyme 1 (UBA1) was sufficient to correct sensory-motor connectivity in the spinal cord of mice with spinal muscular atrophy. Aminoacyl-tRNA synthetases, including GARS, were identified as downstream targets of UBA1. Regulation of GARS by UBA1 occurred via a non-canonical pathway independent of ubiquitylation. Dysregulation of UBA1/GARS pathways in spinal muscular atrophy mice disrupted sensory neuron fate, phenocopying GARS-dependent defects associated with Charcot-Marie-Tooth disease. Sensory neuron fate was corrected following restoration of UBA1 expression and UBA1/GARS pathways in spinal muscular atrophy mice. We conclude that defective sensory motor connectivity in spinal muscular atrophy results from perturbations in a UBA1/GARS pathway that modulates sensory neuron fate, thereby highlighting significant molecular and phenotypic overlap between spinal muscular atrophy and Charcot-Marie-Tooth disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/brain/awy237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158753PMC
October 2018

The many disguises of the signalling endosome.

FEBS Lett 2018 11 17;592(21):3615-3632. Epub 2018 Sep 17.

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, UK.

Neurons are highly complex and polarised cells that must overcome a series of logistic challenges to maintain homeostasis across their morphological domains. A very clear example is the propagation of neurotrophic signalling from distal axons, where target-released neurotrophins bind to their receptors and initiate signalling, towards the cell body, where nuclear and cytosolic responses are integrated. The mechanisms of propagation of neurotrophic signalling have been extensively studied and, eventually, the model of a 'signalling endosome', transporting activated receptors and associated complexes, has emerged. Nevertheless, the exact nature of this organelle remains elusive. In this Review, we examine the evidence for the retrograde transport of neurotrophins and their receptors in endosomes, outline some of their diverse physiological and pathological roles, and discuss the main interactors, morphological features and trafficking destinations of a highly flexible endosomal signalling organelle with multiple molecular signatures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.13235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282995PMC
November 2018

Inhibiting p38 MAPK alpha rescues axonal retrograde transport defects in a mouse model of ALS.

Cell Death Dis 2018 05 22;9(6):596. Epub 2018 May 22.

Department of Neuromuscular Disorders, UCL Institute of Neurology, University College London, London, WC1N 3BG, UK.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the degeneration of upper and lower motor neurons. Defects in axonal transport have been observed pre-symptomatically in the SOD1 mouse model of ALS, and have been proposed to play a role in motor neuron degeneration as well as in other pathologies of the nervous system, such as Alzheimer's disease and hereditary neuropathies. In this study, we screen a library of small-molecule kinase inhibitors towards the identification of pharmacological enhancers of the axonal retrograde transport of signalling endosomes, which might be used to normalise the rate of this process in diseased neurons. Inhibitors of p38 mitogen-activated protein kinases (p38 MAPK) were identified in this screen and were found to correct deficits in axonal retrograde transport of signalling endosomes in cultured primary SOD1 motor neurons. In vitro knockdown experiments revealed that the alpha isoform of p38 MAPK (p38 MAPKα) was the sole isoform responsible for SOD1-induced transport deficits. Furthermore, we found that acute treatment with p38 MAPKα inhibitors restored the physiological rate of axonal retrograde transport in vivo in early symptomatic SOD1 mice. Our findings demonstrate the pathogenic effect of p38 MAPKα on axonal retrograde transport and identify a potential therapeutic strategy for ALS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-018-0624-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964181PMC
May 2018

Functional imaging in microfluidic chambers reveals sensory neuron sensitivity is differentially regulated between neuronal regions.

Pain 2018 Jul;159(7):1413-1425

Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, London, United Kingdom.

Primary afferent sensory neurons are incredibly long cells, often traversing distances of over 1 m in humans. Cutaneous sensory stimuli are transduced in the periphery by specialised end organs or free nerve endings, which code the stimulus into electrical action potentials that propagate towards the central nervous system. Despite significant advances in our knowledge of sensory neuron physiology and ion channel expression, many commonly used techniques fail to accurately model the primary afferent neuron in its entirety. In vitro experiments often focus on the cell somata and neglect the fundamental processes of peripheral stimulus transduction and action potential propagation. Despite this, these experiments are commonly used as a model for cellular investigations of the receptive terminals. We demonstrate that ratiometric calcium imaging performed in compartmentalised sensory neuron cultures can be used to directly and accurately compare the sensitivity and functional protein expression of isolated neuronal regions in vitro. Using microfluidic chambers, we demonstrate that the nerve terminals of cultured dorsal root ganglion neurons can be depolarised to induce action potential propagation, which has both tetrodotoxin-resistant and tetrodotoxin-sensitive components. Furthermore, we show that there is a differential regulation of proton sensitivity between the sensory terminals and somata in cultured sensory neurons. We also demonstrate that capsaicin sensitivity is highly dependent on embryonic dissection age. This approach enables a comprehensive method to study the excitability and regional sensitivity of cultured sensory neurons on a single-cell level. Examination of the sensory terminals is crucial to further understand the properties and diversity of dorsal root ganglion sensory neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/j.pain.0000000000001145DOI Listing
July 2018

Compartmentalized Signaling in Neurons: From Cell Biology to Neuroscience.

Neuron 2017 Nov;96(3):667-679

Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel. Electronic address:

Neurons are the largest known cells, with complex and highly polarized morphologies. As such, neuronal signaling is highly compartmentalized, requiring sophisticated transfer mechanisms to convey and integrate information within and between sub-neuronal compartments. Here, we survey different modes of compartmentalized signaling in neurons, highlighting examples wherein the fundamental cell biological processes of protein synthesis and degradation, membrane trafficking, and organelle transport are employed to enable the encoding and integration of information, locally and globally within a neuron. Comparisons to other cell types indicate that neurons accentuate widely shared mechanisms, providing invaluable models for the compartmentalization and transfer mechanisms required and used by most eukaryotic cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2017.10.015DOI Listing
November 2017