scholarly journals Disease-associated mutations in human BICD2 hyperactivate motility of dynein–dynactin

2017 ◽  
Vol 216 (10) ◽  
pp. 3051-3060 ◽  
Author(s):  
Walter Huynh ◽  
Ronald D. Vale
Keyword(s):  
Spinal Muscular Atrophy ◽  
Hippocampal Neurons ◽  
Muscular Atrophy ◽  
Membrane Vesicle ◽  
Point Mutations ◽  
Neurite Growth ◽  
Retrograde Transport ◽  
Organelle Transport ◽  
Dominant Form

Bicaudal D2 (BICD2) joins dynein with dynactin into a ternary complex (termed DDB) capable of processive movement. Point mutations in the BICD2 gene have been identified in patients with a dominant form of spinal muscular atrophy, but how these mutations cause disease is unknown. To investigate this question, we have developed in vitro motility assays with purified DDB and BICD2’s membrane vesicle partner, the GTPase Rab6a. Rab6a–GTP, either in solution or bound to artificial liposomes, released BICD2 from an autoinhibited state and promoted robust dynein–dynactin transport. In these assays, BICD2 mutants showed an enhanced ability to form motile DDB complexes. Increased retrograde transport by BICD2 mutants also was observed in cells using an inducible organelle transport assay. When overexpressed in rat hippocampal neurons, the hyperactive BICD2 mutants decreased neurite growth. Our results reveal that dominant mutations in BICD2 hyperactivate DDB motility and suggest that an imbalance of minus versus plus end–directed microtubule motility in neurons may underlie spinal muscular atrophy.

scholarly journals Disease-Associated Mutations in Human BICD2 Hyperactivate Motility of Dynein-Dynactin

2017 ◽  
Author(s):  
Walter Huynh ◽  
Ronald D. Vale
Keyword(s):  
Spinal Muscular Atrophy ◽  
Hippocampal Neurons ◽  
Muscular Atrophy ◽  
Membrane Vesicle ◽  
Point Mutations ◽  
Neurite Growth ◽  
Retrograde Transport ◽  
Organelle Transport ◽  
Dominant Form

AbstractBicaudal D2 (BICD2) joins dynein with dynactin into a ternary complex (termed DDB) capable of processive movement. Point mutations in the BICD2 gene have been identified in patients with a dominant form of spinal muscular atrophy, but how these mutations cause disease is unknown. To investigate this question, we have developed in vitro motility assays with purified DDB and BICD2’s membrane vesicle partner, the GTPase Rab6a. Rab6a-GTP, either in solution or bound to artificial liposomes, released BICD2 from an autoinhibited state and promoted robust dynein-dynactin transport. In these assays, BICD2 mutants showed an enhanced ability to form motile DDB complexes. Increased retrograde transport by BICD2 mutants also was observed in cells using an inducible organelle transport assay. When overexpressed in rat hippocampal neurons, the hyperactive BICD2 mutants decreased neurite growth. Our results reveal that dominant mutations in BICD2 hyperactivate DDB motility and suggest that an imbalance of minus- versus plus-end-directed microtubule motility in neurons may underlie spinal muscular atrophy.

scholarly journals Neurite Growth and Polarization on Vitronectin Substrate after in Vitro Trauma is not Enhanced after IGF Treatment

2018 ◽  
Vol 8 (8) ◽  
pp. 151 ◽  
Author(s):  
K. Bergen ◽  
M. Frödin ◽  
C. von Gertten ◽  
A. Sandberg-Nordqvist ◽  
M. Sköld
Keyword(s):  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Brain Injuries ◽  
Neurite Growth ◽  
Cellular Growth ◽  
Neurotrophic Activity ◽  
Igf Binding ◽  
And Migration ◽  
Igf Binding Protein

Following traumatic brain injuries (TBI), insulin-like growth factor (IGF) is cortically widely upregulated. This upregulation has a potential role in the recovery of neuronal tissue, plasticity, and neurotrophic activity, though the molecular mechanisms involved in IGF regulation and the exact role of IGF after TBI remain unclear. Vitronectin (VN), an extracellular matrix (ECM) molecule, has recently been shown to be of importance for IGF-mediated cellular growth and migration. Since VN is downregulated after TBI, we hypothesized that insufficient VN levels after TBI impairs the potential beneficial activity of IGF. To test if vitronectin and IGF-1/IGFBP-2 could contribute to neurite growth, we cultured hippocampal neurons on ± vitronectin-coated coverslips and them treated with ± IGF-1/IGF binding protein 2 (IGFBP-2). Under same conditions, cell cultures were also subjected to in vitro trauma to investigate differences in the posttraumatic regenerative capacity with ± vitronectin-coated coverslips and with ± IGF-1/IGFBP-2 treatment. In both the control and trauma situations, hippocampal neurons showed a stronger growth pattern on vitronectin than on the control substrate. Surprisingly, the addition of IGF-1/IGFBP-2 showed a decrease in neurite growth. Since neurite growth was measured as the number of neurites per area, we hypothesized that IGF-1/IGFBP-2 contributes to the polarization of neurons and thus induced a less dense neurite network after IGF-1/IGFBP-2 treatment. This hypothesis could not be confirmed and we therefore conclude that vitronectin has a positive effect on neurite growth in vitro both under normal conditions and after trauma, but that addition of IGF-1/IGFBP-2 does not have a positive additive effect.

Organelle motility and metabolism in axons vs dendrites of cultured hippocampal neurons

1996 ◽  
Vol 109 (5) ◽  
pp. 971-980 ◽  
Author(s):  
C.C. Overly ◽  
H.I. Rieff ◽  
P.J. Hollenbeck
Keyword(s):  
Regional Variation ◽  
Hippocampal Neurons ◽  
Retrograde Transport ◽  
Organelle Transport ◽  
Microtubule Polarity ◽  
Large Phase ◽  
Organelle Motility ◽  
The Difference ◽  
Metabolic Properties ◽  
Cultured Hippocampal Neurons

Regional regulation of organelle transport seems likely to play an important role in establishing and maintaining distinct axonal and dendritic domains in neurons, and in managing differences in local metabolic demands. In addition, known differences in microtubule polarity and organization between axons and dendrites along with the directional selectivity of microtubule-based motor proteins suggest that patterns of organelle transport may differ in these two process types. To test this hypothesis, we compared the patterns of movement of different organelle classes in axons and different dendritic regions of cultured embryonic rat hippocampal neurons. We first examined the net direction of organelle transport in axons, proximal dendrites and distal dendrites by video-enhanced phase-contrast microscopy. We found significant regional variation in the net transport of large phase-dense vesicular organelles: they exhibited net retrograde transport in axons and distal dendrites, whereas they moved equally in both directions in proximal dendrites. No significant regional variation was found in the net transport of mitochondria or macropinosomes. Analysis of individual organelle motility revealed three additional differences in organelle transport between the two process types. First, in addition to the difference in net transport direction, the large phase-dense organelles exhibited more persistent changes in direction in proximal dendrites where microtubule polarity is mixed than in axons where microtubule polarity is uniform. Second, while the net direction of mitochondrial transport was similar in both processes, twice as many mitochondria were motile in axons than in dendrites. Third, the mean excursion length of moving mitochondria was significantly longer in axons than in dendrites. To determine whether there were regional differences in metabolic activity that might account for these motility differences, we labeled mitochondria with the vital dye, JC-1, which reveals differences in mitochondrial transmembrane potential. Staining of neurons with this dye revealed a greater proportion of highly charged, more metabolically active, mitochondria in dendrites than in axons. Together, our data reveal differences in organelle motility and metabolic properties in axons and dendrites of cultured hippocampal neurons.

ASMN2Splicing Modifier Rescues the Disease Phenotypes in an In Vitro Human Spinal Muscular Atrophy Model

2019 ◽  
Vol 28 (7) ◽  
pp. 438-453 ◽  
Author(s):  
Ye Seul Son ◽  
Kwangman Choi ◽  
Hana Lee ◽  
Ohman Kwon ◽  
Kwang Bo Jung ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Disease Phenotypes

SMN1 gene point mutations in type I–IV proximal spinal muscular atrophy patients with a single copy of SMN1

2015 ◽  
Vol 51 (9) ◽  
pp. 925-931
Author(s):  
V. V. Zabnenkova ◽  
E. L. Dadali ◽  
S. B. Artemieva ◽  
I. V. Sharkova ◽  
G. E. Rudenskaya ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Point Mutations ◽  
Single Copy ◽  
Type I ◽  
Smn1 Gene ◽  
Proximal Spinal Muscular Atrophy

Novel point mutations in survival motor neuron 1 gene expand the spectrum of phenotypes observed in spinal muscular atrophy patients

2014 ◽  
Vol 24 (7) ◽  
pp. 617-623 ◽  
Author(s):  
Maria Jędrzejowska ◽  
Monika Gos ◽  
Janusz G. Zimowski ◽  
Anna Kostera-Pruszczyk ◽  
Barbara Ryniewicz ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Muscular Atrophy ◽  
Point Mutations ◽  
Survival Motor Neuron

scholarly journals Polarized localization of voltage-gated Na+ channels is regulated by concerted FGF13 and FGF14 action

2016 ◽  
Vol 113 (19) ◽  
pp. E2665-E2674 ◽  
Author(s):  
Juan Lorenzo Pablo ◽  
Chaojian Wang ◽  
Matthew M. Presby ◽  
Geoffrey S. Pitt
Keyword(s):  
Action Potential ◽  
Hippocampal Neurons ◽  
Single Point ◽  
Point Mutations ◽  
Surface Expression ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Action Potential Initiation ◽  
Potential Initiation

Clustering of voltage-gated sodium channels (VGSCs) within the neuronal axon initial segment (AIS) is critical for efficient action potential initiation. Although initially inserted into both somatodendritic and axonal membranes, VGSCs are concentrated within the axon through mechanisms that include preferential axonal targeting and selective somatodendritic endocytosis. How the endocytic machinery specifically targets somatic VGSCs is unknown. Here, using knockdown strategies, we show that noncanonical FGF13 binds directly to VGSCs in hippocampal neurons to limit their somatodendritic surface expression, although exerting little effect on VGSCs within the AIS. In contrast, homologous FGF14, which is highly concentrated in the proximal axon, binds directly to VGSCs to promote their axonal localization. Single-point mutations in FGF13 or FGF14 abrogating VGSC interaction in vitro cannot support these specific functions in neurons. Thus, our data show how the concerted actions of FGF13 and FGF14 regulate the polarized localization of VGSCs that supports efficient action potential initiation.

scholarly journals Chondrolectin affects cell survival and neuronal outgrowth in in vitro and in vivo models of spinal muscular atrophy

2013 ◽  
Vol 23 (4) ◽  
pp. 855-869 ◽  
Author(s):  
James N. Sleigh ◽  
Antón Barreiro-Iglesias ◽  
Peter L. Oliver ◽  
Angeliki Biba ◽  
Thomas Becker ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Cell Survival ◽  
Muscular Atrophy ◽  
In Vivo Models ◽  
Neuronal Outgrowth

scholarly journals Synthesis and Characterization of Pseudocantharidins, Novel Phosphatase Modulators That Promote the Inclusion of Exon 7 into the SMN (Survival of Motoneuron) pre-mRNA

2011 ◽  
Vol 286 (12) ◽  
pp. 10126-10136 ◽  
Author(s):  
Zhaiyi Zhang ◽  
Olga Kelemen ◽  
Maria A. van Santen ◽  
Sharon M. Yelton ◽  
Alison E. Wendlandt ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Central Element ◽  
Detectable Effect ◽  
Splice Site Selection ◽  
Eukaryotic Gene ◽  
Constitutive Splicing ◽  
New Compounds

Alternative pre-mRNA splicing is a central element of eukaryotic gene expression. Its deregulation can lead to disease, and methods to change splice site selection are developed as potential therapies. Spinal muscular atrophy is caused by the loss of the SMN1 (survival of motoneuron 1) gene. A therapeutic avenue for spinal muscular atrophy treatment is to promote exon 7 inclusion of the almost identical SMN2 (survival of motoneuron 2) gene. The splicing factor tra2-beta1 promotes inclusion of this exon and is antagonized by protein phosphatase (PP) 1. To identify new compounds that promote exon 7 inclusion, we synthesized analogs of cantharidin, an inhibitor of PP1, and PP2A. Three classes of compounds emerged from these studies. The first class blocks PP1 and PP2A activity, blocks constitutive splicing in vitro, and promotes exon 7 inclusion in vivo. The second class has no measurable effect on PP1 activity but activates PP2A. This class represents the first compounds described with these properties. These compounds cause a dephosphorylation of Thr-33 of tra2-beta1, which promotes exon 7 inclusion. The third class had no detectable effect on phosphatase activity and could promote exon 7 via allosteric effects. Our data show that subtle changes in similar compounds can turn a phosphatase inhibitor into an activator. These chemically related compounds influence alternative splicing by distinct mechanisms.

scholarly journals Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1636 ◽  
Author(s):  
Chris D. Balak ◽  
Jesse M. Hunter ◽  
Mary E. Ahearn ◽  
David Wiley ◽  
Gennaro D'urso ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Diagnostic Assays ◽  
Missense Variants ◽  
Thioester Bond ◽  
In The Wild ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

Background: X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 (UBA1). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the UBA1 gene, which contains the active adenylation domain (AAD). Methods: In this study, our group characterized the three known missense variants in vitro. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions in vitro to determine possible biochemical effects of the missense variants. Results: Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. Conclusions: Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1’s enzymatic activity with Ubiquitin, to other roles such as altering UBA1 mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important in vivo binding partners.  These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations’ biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.

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