Neurofilaments are space-filling cytoskeletal polymers that function to increase the cross-sectional area of axons. These polymers are transported along axons at fast rates, but the overall rate is slow because the movements are interrupted by prolonged pauses. Neurofilaments accumulate in axons during the growth of axonal caliber and they also accumulate abnormally and excessively in a wide range of neurodegenerative diseases, most notably amyotrophic lateral sclerosis. Mutations that disrupt neurofilament assembly also cause one form of Charcot-Marie-Tooth disease. The long-term goal of our research is to understand the transport and assembly dynamics of neurofilaments in axons and the mechanisms that cause neurofilaments to accumulate in development and disease. We propose three specific aims.
Aim 1 is to investigate the assembly dynamics of neurofilaments in axons. Axonal neurofilaments can be 100 ?m or more in length in vivo and they can also exchange subunits with a soluble pool, but little is known about how these processes occur. We propose that neurofilaments can lengthen by end-to-end annealing of pre-formed filaments and that they can exchange subunits by addition and loss of subunits along the length of the filaments, a process that we term intercalary subunit exchange. We will use cell fusion as well as photobleaching and photoactivation strategies to test these hypotheses in cultured neurons. We will also extend our studies to other types of intermediate filament proteins to establish whether our findings are more generally applicable.
Aim 2 is to investigate the role of phosphorylation in neurofilament transport. We propose that phosphorylation of neurofilaments by CDK5 and ERK1/2 kinases regulates their transport in an additive and dose-dependent manner by increasing the proportion of the time the neurofilaments spend pausing. We will test these hypotheses in cultured neurons by using site-directed mutagenesis to mimic phosphorylated or non- phosphorylated states at specific sites. We will also manipulate kinase activities directly using pharmacological inhibitors and constitutively active or dominant negative kinase constructs.
Aim 3 is to investigate the functional significance of neurofilament pausing. We propose that neurofilament pausing is a critical determinant of axonal neurofilament content. We will test the hypothesis that neurofilament phosphorylation causes axonal neurofilaments to accumulate by increasing their residence time in axons. We will also use long-term myelinating cultures to test the hypothesis that myelinating cells locally slow neurofilament transport by increasing the proportion of the time that they spend pausing. The use of live-cell imaging to study neurofilament transport in myelinating axons in culture is a particularly innovative aspect of this Aim. The mechanisms that regulate neurofilament pausing are likely targets for disease processes that lead to excessive accumulation of neurofilaments in axons.

Public Health Relevance

Neurofilaments are of clinical interest because they accumulate excessively in nerve cells in a wide range of debilitating neurological disorders including motor neuron disease, giant axonal neuropathy, Charcot-Marie- Tooth disease, Alzheimer's disease, Parkinson's disease, diabetic neuropathy and spinal muscular atrophy. These accumulations exacerbate the disease progression by disrupting the movement of other axonal components which are essential for normal function. Our studies on neurofilament assembly and movement in axons will illuminate the basic mechanisms that can cause neurofilaments to accumulate in axons and may ultimately lead to the development of therapeutic strategies to alleviate these cytoskeletal log-jams, thereby delaying the progression of these tragic diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS038526-15
Application #
8402823
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Gubitz, Amelie
Project Start
1999-04-01
Project End
2014-12-31
Budget Start
2013-01-01
Budget End
2014-12-31
Support Year
15
Fiscal Year
2013
Total Cost
$310,308
Indirect Cost
$103,436
Name
Ohio State University
Department
Neurosciences
Type
Schools of Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Monsma, Paula C; Li, Yinyun; Fenn, J Daniel et al. (2014) Local regulation of neurofilament transport by myelinating cells. J Neurosci 34:2979-88
Brown, Anthony; Jung, Peter (2013) A critical reevaluation of the stationary axonal cytoskeleton hypothesis. Cytoskeleton (Hoboken) 70:1-11
Uchida, Atsuko; Colakoglu, Gulsen; Wang, Lina et al. (2013) Severing and end-to-end annealing of neurofilaments in neurons. Proc Natl Acad Sci U S A 110:E2696-705
Li, Yinyun; Jung, Peter; Brown, Anthony (2012) Axonal transport of neurofilaments: a single population of intermittently moving polymers. J Neurosci 32:746-58
Yuan, Liang; Zheng, Yuan F; Zhu, Junda et al. (2012) Object tracking with particle filtering in fluorescence microscopy images: application to the motion of neurofilaments in axons. IEEE Trans Med Imaging 31:117-30
Colakoglu, Gulsen; Brown, Anthony (2009) Intermediate filaments exchange subunits along their length and elongate by end-to-end annealing. J Cell Biol 185:769-77
Alami, Nael H; Jung, Peter; Brown, Anthony (2009) Myosin Va increases the efficiency of neurofilament transport by decreasing the duration of long-term pauses. J Neurosci 29:6625-34
Uchida, Atsuko; Alami, Nael H; Brown, Anthony (2009) Tight functional coupling of kinesin-1A and dynein motors in the bidirectional transport of neurofilaments. Mol Biol Cell 20:4997-5006
Yan, Yanping; Jensen, Kitty; Brown, Anthony (2007) The polypeptide composition of moving and stationary neurofilaments in cultured sympathetic neurons. Cell Motil Cytoskeleton 64:299-309
Trivedi, Niraj; Jung, Peter; Brown, Anthony (2007) Neurofilaments switch between distinct mobile and stationary states during their transport along axons. J Neurosci 27:507-16

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