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The Genetics of Axonal Transport and Axonal Transport Disorders


Abstract and Figures

Neurons are specialized cells with a complex architecture that includes elaborate dendritic branches and a long, narrow axon that extends from the cell body to the synaptic terminal. The organized transport of essential biological materials throughout the neuron is required to support its growth, function, and viability. In this review, we focus on insights that have emerged from the genetic analysis of long-distance axonal transport between the cell body and the synaptic terminal. We also discuss recent genetic evidence that supports the hypothesis that disruptions in axonal transport may cause or dramatically contribute to neurodegenerative diseases.
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The Genetics of Axonal Transport
and Axonal Transport Disorders
Jason E. Duncan, Lawrence S. B. Goldstein
eurons are specialized cells with a complex
architecture that includes elaborate dendritic
branches and a long, narrow axon that extends from
the cell body to the synaptic terminal. The organized
transport of essential biological materials throughout the
neuron is required to support its growth, function, and
viability. In this review, we focus on insights that have
emerged from the genetic analysis of long-distance axonal
transport between the cell body and the synaptic terminal.
We also discuss recent genetic evidence that supports the
hypothesis that disruptions in axonal transport may cause or
dramatically contribute to neurodegenerative diseases.
The axon of a neuron conducts the transmission of action
potentials from the cell body to the synapse. The axon also
provides a physical conduit for the transport of essential
biological materials between the cell body and the synapse
that are required for the function and viability of the neuron.
A diverse array of cargoes including membranous organelles,
synaptic vesicle precursors, signaling molecules, growth
factors, protein complexes, cytoskeletal components, and
even the sodium and potassium channels required for action
potential propagation are actively transported from their site
of synthesis in the cell body through the axoplasm to
intracellular target sites in the axon and synapse.
Simultaneously, neurotrophic signals are transported from
the synapse back to the cell body to monitor the integrity of
target innervation. The length of axons in the peripheral
nervous system can be in excess of one meter in humans, and
even longer in larger animals, making these cells particularly
reliant on the efficient and coordinated physical transport of
materials through the axons for their function and viability.
The length and narrow caliber of axons coupled with the
amount of material that must be transported raises the
possibility that this system might exhibit significant
vulnerability to perturbation. It has been proposed that
disruptions in axonal transport may lead to axonal transport
defects that manifest as a number of different
neurodegenerative diseases [1]. In this review, we focus on the
use of genetics to understand axonal transport, including the
identification and functional characterization of components
required for axonal transport, and the biological and medical
consequences when these functions are compromised.
Basic Features of the Axonal Transport System
Simplistically, the axonal transport system comprises cargo,
motor proteins that power cargo transport, cytoskeletal
filaments or ‘‘ tracks’’ along which the motors generate force
and movement, linker proteins that attach motor proteins to
cargo or other cellular structures, and accessory molecules
that initiate and regulate transport. Defective axonal
transport and neurodegenerative diseases could potentially
result from disruptions in any of the components required
for axonal transport.
Long-distance transport in the axon is primarily a
microtubule-dependent process. The microtubule tracks
within an axon possess inherent polarity and are uniformly
oriented with the fast-growing (plus) ends projecting toward
the synapse and the slow-growing (minus) ends toward the
cell body [2]. The motor proteins that power axonal transport
on microtubules are members of the kinesin and cytoplasmic
dynein superfamilies. Kinesins are generally plus-end–
directed motor proteins that transport cargoes such as
synaptic vesicle precursors and membranous organelles
anterogradely toward the synapse (Figure 1). Cytoplasmic
dyneins are minus-end–directed motor proteins that
transport cargoes including neurotrophic signals, endosomes,
and other organelles and vesicles retrogradely toward the cell
body (Figure 1). Retrograde transport may not be exclusive to
dyneins, however, as a few kinesins that translocate cargo in
the retrograde direction have been identified [3,4]. In
mammals, the kinesin superfamily consists of approximately
45 members (KIFs) grouped into 14 subfamilies (reviewed in
[5]). Kinesins comprise one to four motor polypeptides called
heavy chains that contain a highly conserved motor domain,
with ATPase and microtubule-binding regions, and a
Editor: Elizabeth M. C. Fisher, University College London, United Kingdom
Citation: Duncan JE, Goldstein LSB (2006) The genetics of axonal transport and
axonal transport disorders. PLoS Genet 2(9): e124. DOI: 10.1371/journal.pgen.
DOI: 10.1371/journal.pgen.0020124
Copyright: Ó 2006 Duncan and Goldstein. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Abbreviations: AchE, acetylcholinesterase; AD, Alzheimer disease; ALS,
amyotrophic lateral sclerosis; APP, amyloid precursor protein; ChAT, choline
acetyltransferase; CMT, Charcot-Marie-Tooth disease; CSP, cysteine-string protein;
Dhc, cytoplasmic dynein heavy cha in; Dlc, cytoplasmic dynein light chain; Dync1h1,
dynein heavy chain gene; GAP-43, growth associated protein 43; GSK 3b, glycogen
synthase kinase 3 b; HAP1, Huntingtin-associated protein 1; HD, Huntington
disease; HSP, Hereditary Spastic Paraplegia; HSP(SPG 10), Hereditary Spastic
Paraplegia Type 10; Htt, huntingtin protein; JIP, JNK interacting protein; JNK, cJun
-terminal kinase; Khc, kinesin heavy chain; KIFs, kinesin superfamily members;
Klc, kinesin light chain; PS1, presenilin-1; Snb-GFP, synaptobrevin-GFP; SOD1, Cu/
Zn superoxide dismutase; Syt, synaptotagmin; UNC-104, UNCoordinated-104
Jason E. Duncan and Lawrence S. B. Goldstein are from the Howard Hughes
Medical Institute, Department of Cellular and Molecular Medicine, School of
Medicine, University of California San Diego, La Jolla, California, United States of
America. E-mail: (JED); lgoldste (LSBG)
PLoS Genetics | September 2006 | Volume 2 | Issue 9 | e1241275
divergent tail domain. Regulatory and/or accessory subunits,
such as the kinesin light chain (Klc), are thought to interact
with the tail domain of the kinesin heavy chain (Khc) to
confer cargo-binding specificity and regulation (Figure 1)
(reviewed in [6]). In contrast to kinesin, the cytoplasmic
dynein family in mammals is much smaller, consisting of only
two members. Cytoplasmic dynein, however, is a larger and
more complex microtubule motor, comprising two dynein
heavy chain (Dhc) motor subunits and various intermediate,
light intermediate, and light chain (Dlc) subunits (Figure 1)
(reviewed in [7]). Cytoplasmic dynein appears to employ a
‘‘ subunit heterogeneity’’ approach to support a wide range of
essential cellular functions with only a few copies of the
cytoplasmic dynein motor peptide and a diverse array of
dynein-associated accessory proteins that impart cargo-
binding specificity and functional activity [6,8]. Considerable
evidence suggests that dynein function is dependent on an
equally large protein complex called dynactin, which is
proposed to link cytoplasmic dynein to its cargo and/or to
increase dynein processivity through an association with
microtubules (Figure 1) [9,10].
Based on the kinetics of transport determined from classic
pulse-chase labeling experiments, axonal transport is
classified as either fast or slow (reviewed in [11,12]). Fast
axonal transport occurs in both the retrograde and
anterograde directions at a rate of 0.5–10 lm/sec and includes
the transport of membrane-bound organelles, mitochondria,
neurotransmitters, channel proteins, multivesicular bodies,
and endosomes. In contrast, slow axonal transport occurs in
the anterograde direction at a rate of 0.01–0.001 lm/sec,
considerably slower than fast axonal transport [12].
Cytoskeletal components, such as neurofilaments, tubulin,
and actin, as well as proteins such as clathrin and cytosolic
enzymes are transported at this slower rate [12]. Current
thought is that slow axonal transport is mediated by the same
microtubule motors that participate in fast axonal transport,
with fast instantaneous transport of cargo interspersed with
prolonged pauses [13–15].
Mutations Disrupting Motor Proteins
Classic studies using extruded squid axoplasm identified
kinesin and cytoplasmic dynein as candidate motors required
for axonal transport [16–20]. Since then, many different
animal model systems have been used to genetically investigate
axonal transport mechanisms. Such studies reveal
considerable diversity in kinesin function in the axon (Table 1).
The requirement for conventional kinesin (Kinesin-1) in
axonal transport was revealed in Drosophila melanogaster larvae
with lesions in Khc and Klc genes. These mutants exhibit
axonal swellings containing accumulations of transported
vesicles, synaptic membranes, and mitochondria [21–23].
Such axonal ‘‘ organelle jams’’ are a phenotypic hallmark of
compromised axonal transport and result in a posterior
paralysis of mutant larvae. Loss of function of the neuronal
Figure 1. Cytoplasmic Dynein and Kinesin Power Axonal Transport
Schematic diagram of the microtubule motor proteins cytoplasmic dynein and kinesin. Cytoplasmic dynein transports cargo in the retrograde direction
toward the minus ends of microtubules whereas kinesin transports cargo in the anterograde direction toward the plus ends. Cytoplasmic dynein is a
large multimeric protein complex comprising two heavy chain subunits (red) that possess microtubule binding and ATPase activity, two intermediate
chains (yellow), two light intermediate chains (indigo), and an assortment of light chains (light pink, green, orange) (reviewed in [7]). Dynactin, a large
multisubunit protein complex of comparable size to cytoplasmic dynein, is proposed to link the dynein motor to cargo and/or increases its processivity.
The largest dynactin subunit, p150
(turquoise), forms an elongated dimer that interacts with the dynein intermediate chain and binds to
microtubules via a highly conserved CAP-Gly motif at the tip of globular heads. The dynactin subunit p50 (dark pink) occupies a central position linking
to cargo. The conventional kinesin holoenzyme, also known as kinesin-1, is a heterotetramer comprising two Khc subunits (red) with
microtubule binding and ATPase domains, a central coiled stalk, and a tail domain that interacts with two Klc subunits (green). Klcs may mediate cargo-
binding via an intermediate scaffold protein (blue) that binds a cargo transmembrane protein (yellow).
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Table 1. Kinesin Genes Required for Axonal Transport
Gene Common Name Organism Lesion Phenotype/Disease Inferred Function Accession
for mRNA
for Protein
Kinesin-1 Kif5A Kinesin heavy chain H. sapiens Motor domain N256S,
microtubule binding
domain R280C
Hereditary Spastic
Paraplegia SPG10
Unknown NM_004984 NP_004975 [24,25]
Kif5A Kinesin heavy chain M. musculus Null, conditional knockout Loss of large caliber axons,
neurofilament accumulation
in cell bodies of peripheral
sensory neurons
Slow axonal transport
of neurofilaments
NM_008447 NP_032473 [15]
Kif5B Kinesin heavy chain M. musculus Null Impaired mitochondrial and
lysosomal dispersion
Mitochondrial transport NM_008448 NP_032474 [27]
Kif5C Kinesin heavy chain M. musculus Null Viable, decrease in motor
neurons, reduced brain size
NM_008449 NM_032475 [28]
Khc Kinesin heavy chain D. melanogaster Khc
, Khc
Axonal swellings containing
vesicles, mitochondria,
and organelles
Anterograde axonal
NM_057242 NP_476590 [22,23]
unc-116 Kinesin heavy chain C. elegans e2281 Mislocalization of synaptic
vesicles, Jip3
NM_066441 NP_498842 [48]
KLC1 Kinesin light chain M. musculus Knockout Impaired axonal transport of APP,
b-secretase, PS1, GAP-43,
synapsin 1 and Trk-A
Anterograde axonal
AFO55665 AAC27740 [69,70]
Klc Kinesin light chain D. melanogaster Klc
,Df(3L)8ex94 Synaptic vesicle accumulation Anterograde axonal
NM_079325 NP_524049 [21]
Klc2 Kinesin light chain C. elegans km11, km28 Mislocalization of Snb-GFP Synaptic vesicle transport AAK52182
Klc Kinesin light chain Cell culture HA-KLC TPRs, HA-KLC-176 Mislocalization of JIP2, JIP3 Interaction with scaffold
Kinesin-2 Kif3A/3B Heterotrimeric
M. musculus Antibody microinjection Reduced fast axonal transport Axonal transport of fodrin
associating vesicles
D. melanogaster KLP64D
, siRNA,
, siRNA
Axonal accumulation of AchE
and ChAT
Axonal transport of AchE
and ChAT
Kinesin-3 Kif1A Monomeric Kinesin M. musculus Knockout Decreased axonal transport
of synaptic vesicles
Synaptic vesicle transport NM_008440 NP_003246 [30,31]
unc-104 Monomeric Kinesin C. elegans 17 alleles including
rh443, rh142
Paralyzed locomotion, poor
Synaptic vesicle transport NM_171017 NP_741019 [29]
Kif1B b Monomeric Kinesin M. musculus Knockout Reduced axonal transport
of Syt, SV2
Synaptic vesicle transport NM_207682
Kif1B b Monomeric Kinesin H. sapiens ATP binding domain Q98L Charcot-Marie-Tooth
Disease Type 2A1
Synaptic vesicle transport [32]
Knockout or mutagenic lesions in the listed genes that encode protein components of kinesin motors result in defective axonal transport. Accession numbers for each gene were obtained from the National Center for Biotechnology Information (NCBI)
database ( accessed 2 May 2006.
Denotes genes that have multiple accession numbers for different isoforms derived from alternative splicing.
DOI: 10.1371/journal.pgen.0020124.t001
PLoS Genetics | September 2006 | Volume 2 | Issue 9 | e1241277
Kinesin-1 family member KIF5A is linked to the human
neurodegenerative disease Hereditary Spastic Paraplegia
(HSP) Type 10 (HSP(SPG10)) [24,25]. HSP is a group of
clinically heterogeneous neurodegenerative disorders
characterized by progressive spasticity and mild weakness of
the lower limbs [26]. Although the mechanistic cause of
HSP(SPG10) remains unclear, the observation that KIF5A is
required for the transport of neurofilaments implies a
possible defect in slow axonal transport in the pathogenesis
of HSP(SPG10) [15]. The ubiquitous Kinesin-1 family member
KIF5B is required for the transport of both mitochondria and
lysosomes [27]. Elucidation of a defined cellular role for
neuronal-specific Kinesin-1 KIF5C is hindered by its
apparent functional redundancy with KIF5A and KIF5B [28].
Members of the Kinesin-3 family, including UNC-104,
KIF1A, and KIF1B, are required for the axonal transport of
specific membrane-bound organelles such as synaptic vesicle
precursors and mitochondria. Mutants of the unc-104 gene of
C. elegans are paralyzed and have fewer synaptic vesicles than
wild-type animals [29]. The subcellular distribution of other
membrane-bound organelles such as the endoplasmic
reticulum, Golgi apparatus, and mitochondria appear normal
in these mutants, supporting the idea that the specific role for
UNC-104 is in the anterograde transport of synaptic vesicle
components [29]. Mice lacking KIF1A, a neuronal-specific
homolog of UNC-104, die shortly after birth and suffer
marked neuronal degeneration associated with a similar
decrease in synaptic vesicle transport and a subsequent
reduction in the density of these vesicles in the nerve
terminals [30]. Fractionation and immunoisolation
experiments revealed that KIF1A associates with a specific
subclass of synaptic vesicles containing synaptotagmin,
synaptophysin, and Rab3A [31]. KIF1Bb associates with yet a
different subclass of synaptic vesicle components that contain
synaptophysin, synaptotagmin, and the synaptic membrane
integral protein SV2 [32]. Interestingly, the human
neurodegenerative disorder Charcot-Marie-Tooth (CMT)
disease Type 2A1, an inherited neuropathy characterized by
weakness and atrophy of distal muscles, is linked to a
mutation in the ATP binding site of the motor domain of
human KIF1Bb [32]. In a KIF1Bb knockout, heterozygous
mice develop multiple nervous-system abnormalities similar
to those observed in UNC-104/KIF1A mutants, including a
decrease in the transport of synaptic vesicle proteins and a
reduction of these vesicles at the synapse [32].
Together these genetic experiments support the hypothesis
that KIFs support various cellular functions by transporting
different classes of organelles and vesicles in axons.
Unlike the kinesin superfamily, in which different members
of a large superfamily support diverse cellular functions,
cytoplasmic dynein comprises an invariant motor subunit
with variations in other protein subunits that potentially alter
motor function and cargo specificity. Consequently, isolating
and interpreting lesions in the cytoplasmic dynein motor has
been difficult since dynein is required for multiple functions
in the neuron, including axonal transport [33,34].
Nonetheless, in vivo evidence supports a role for cytoplasmic
dynein in retrograde axonal transport (Table 2).
Although null mutants die early in development,
hypomorphic alleles of the cytoplasmic Dhc in Drosophila
result in larval paralysis with accumulations of synaptic
vesicle components in axonal swellings that are
indistinguishable from phenotypes observed in Khc mutants
[35]. Hypomorphic mutations in both the C. elegans Dhc and
Dlc genes also caused reduced locomotion in animals and
ectopic accumulation of the synaptic vesicle components
synaptobrevin, synaptotagmin, and the kinesin motor UNC-
104 at the terminal ends of mechanosensory processes [36].
Finally, two mutations in the mouse dynein heavy chain gene
(Dync1h1), Loa and Cra1, cause progressive motor neuron
degeneration in heterozygotes [37]. A marked alteration in
the retrograde transport of a fluorescent tetanus toxin tracer
was observed in cultured motor neurons isolated from Loa
homozygous mice [37]. Although mutant forms of the Dync1h1
gene are ubiquitously expressed in heterozygous mice, the
lesions appear to primarily perturb axonal transport in
motor neurons, indicating that for unknown reasons, motor
neurons are extremely sensitive to alterations in dynein
function [37].
Mutations in Non-Motor Components Disrupt
Axonal Transport
Lesions in kinesin and cytoplasmic dynein disrupt critical
functions in axonal transport, but factors associated with the
motors, such as dynactin, may also be essential for transport
(Table 3). Membrane-bound organelles transported in the
axon often move bidirectionally, alternating between
anterograde and retrograde motion, with net movement in
one direction. This suggests that dynein and kinesin are
present on the same organelles and their activity is
coordinated. One candidate to mediate this coordination is
the dynactin complex [38]. Strong genetic interactions have
been observed between kinesin, cytoplasmic dynein, and the
dynactin complex in Drosophila [35]. Dynactin is also required
for bidirectional transport of lipid droplets in Drosophila
embryos and mediates the interaction between kinesin and
cytoplasmic dynein in Xenopus melanophore cells [39,40].
Consequently, caution must be exercised when interpreting
phenotypes associated with mutations in dynactin
components because both anterograde and retrograde
transport parameters may be affected, as observed in the
axonal transport of mitochondria in Drosophila p150
mutants [41]. In another study, the overexpression of a
dominant negative form of dynactin component p150
Drosophila caused phenotypes similar to those observed in
both Dhc and Khc mutants [35]. Partial loss-of-function of
or overexpression of p50 dynamitin in C. elegans
resulted in ectopic accumulation of synaptic vesicle
components [36]. The overexpression of p50 dynamitin
disrupts the dynactin complex and inhibits cytoplasmic
dynein function, circumventing the difficulty of isolating
viable dynein mutants. The targeted overexpression of p50
dynamitin in mouse motor neurons caused an accumulation
of synaptophysin and aggregation of neurofilaments in axons,
as well as late onset motor neuron degeneration [42].
Although mutant cytoplasmic dynein has yet to be identified
as a causative factor of a human neurological disorder,
dynactin is directly linked to a number of human
neurodegenerative diseases. Lesions in the conserved CAP-
Gly microtubule-binding motif of the p150
subunit of
dynactin have been identified in a family with a heritable
form of motor neuron disease. These individuals exhibit
weakness in the distal limbs, abnormal accumulations of both
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Table 2. Cytoplasmic Dynein and Dynactin Genes Required for Axonal Transport
Cytoplasmic Dynein
or Dynactin Protein
Gene Common
Organism Lesion Phenotype/
Number mRNA
Number Protein
Cytoplasmic dynein
heavy chain
Dync1h1 Legs at odd
angles (Loa1),
Cramping 1 (Cra1)
M. musculus F580Y, Y1055C Impaired retrograde transport
in a motor neurons
axonal transport
NM_030238 NP_084514 [37]
dhc-1 Dynein heavy chain C. elegans js319, js121,
Mislocalization of Snb-GFP,
Syt, UNC-104
Axonal transport
of synaptopbrevin
NM_058962 NP_491363 [36]
cDhc64C Dynein heavy chain D. melanogaster cDhc64C
Mislocalization of Syt, CSP Retrograde
axonal transport
Cytoplasmic dynein,
light intermediate chain
dli-1 Dynein light,
intermediate chain
C. elegans js351, ku266 Mislocalization of Snb-GFP,
Syt, UNC-104
Component of
dynein complex
NM_070117 NP_502518 [36]
Cytoplasmic dynein
light chain
Dynein light chain D. melanogaster robl
Mislocalization of Syt, ChAT,
CSP, Kinesin-I,
and Kinesin-II motors
Modulation of
dynein function
NM_079047 NP_523771 [97]
p150 DCTN1 p150
, dynactin H. sapiens T1249I, M571T,
Sporadic and familial ALS Retrograde
axonal transport
DCTN1 p150
, dynactin H. sapiens G59S Inclusions of dynein and
dynactin in motor neurons,
lower motor neuron disease
axonal transport
[44, 98]
Glued p150
D. melanogaster Overexpression
Larval paralysis,
of Syt in axons
axonal transport
NM_079337 NP_081427 [35]
dnc-1 p150
C. elegans or404ts Mislocalization of Snb-GFP,
Syt, UNC-104
axonal transport
NM_069632 NP_502033 [36]
p50 Dctn2 p50, dynamitin M. musculus Overexpression Impaired retrograde
axonal transport,
motor neuron disease
axonal transport
NM_027151 NP_081427 [42]
dnc-2 p50, dynamitin C. elegans Overexpression Mislocalization of Snb-GFP,
Syt, UNC-104
axonal transport
NM_065885 NP_498286 [36]
Knockout or mutagenic lesions in the listed genes that encode protein components of the cytoplasmic dynein motor or dynactin complex result in defective axonal transport. Accession numbers for each gene were obtained from the National Center
for Biotechnology Information (NCBI) database ( accessed 22 August 2006.
Denotes genes that have multiple accession numbers for different isoforms derived from alternative splicing.
DOI: 10.1371/journal.pgen.0020124.t002
PLoS Genetics | September 2006 | Volume 2 | Issue 9 | e1241279
Table 3. Accessory Genes Required for Axonal Transport
Gene Common
Organism Lesion Phenotype/Disease Inferred
for mRNA
for Protein
Amyloid precursor
Appl APP, APP-like, Appl D. melanogaster Appl
, overexpression of APP695,
Swedish, London, 695APLP2,
Synaptic vesicle accumulations
Linker protein
for Kinesin-1
NM_057278 NP_476626 [71]
App like interacting
Aplip1 Jip1 D. melanogaster ek4 Mislocalization of Syt, impaired
transport of Snb-GFP, mitochondria
Mitochondria, synaptic
vesicle transport
Huntingtin protein dhtt Huntingtin D. melanogaster dhtt RNAi, overexpression of
httex1-93Q, MJD78Q, 108Q,
127Q, MJD77Q-NES
Impaired axonal transport,
accumulations of CSP, Syt
Unknown NM_143372 NP_651629 [76]
htt Huntingtin L. pealii HD548-Q62, HD548-Q100 Impaired anterograde and
retrograde transport
Unknown [79]
Hdh Huntingtin M. musculus HD72 Impaired vesicle and
mitochondria transport
Unknown NM_010414 NP_034544 [77]
Huntingtin associated
hap1 HAP-1 Cell culture siRNA Impaired APP vesicle transport Interaction with Klc
and Htt
Superoxide dismutase 1 Sod1 SOD1 M. musculus G93A Impaired slow axonal transport,
swollen axons with neurofilament
Unknown NM_011434 NP_035564 [84,85]
Sod1 SOD1 M. musculus G37R, G85R Impaired slow axonal transport
of neurofilaments, b-tubulin
Unknown NM_011434 NP_035564 [83]
Sod1 SOD1 M. musculus G93A Inhibition of retrograde transport,
disruption of dynein localization
Unknown NM_011434 NP_035564 [88]
presenilin 1 PS1 Presenilin M. musculus
PS1 knockout, KI
Reduced levels of PS1, APP,
and Syn in sciatic nerve
Modulation of GSK
3b, Kinesin-1
NM_008943 NP_032969 [99]
c Jun-NH2 terminal
Kinase interacting protein 1
Jip1 Jip1 Cell culture Jip1(307–700), Jip1(Y709A) Jip1 mislocalization in neurites Scaffold protein AFO03115 Q9WVI9
c Jun-NH2
terminal Kinase
jnk-1 JNK C. elegans gk1 Mislocalization of Snb-GFP Synaptic vesicle
NM_171371 NP_741434 [48]
c Jun-NH2 terminal
Kinase Kinase
jkk-1 JNK-kinase C. elegans km2 Mislocalization of Snb-GFP Synaptic vesicle
NM_076512 NP_508913 [48]
Sunday driver dSyd Syd, Jip3, JSAP1 D. melanogaster sydZ, sydD1, syd2H Synaptic vesicle accumulations Scaffold protein NM_079913
unc-16 Syd, Jip3, JSAP1 C. elegans n730, ju79, e109, ju146 Mislocalization of Snb-GFP Synaptic vesicle
NM_171221 NP_741263 [48]
UNC-14 unc-14 UNC-14 C. elegans ju56 Mislocalization of Snb-GFP Cargo and regulator
of Kinesin-1
NM_059617 NP_492018 [92]
UNC-76 unc-76 UNC-76 D. melanogaster l(1)G0310 Synaptic vesicle accumulations Integration of
kinesin activity
Milton milt Milton D. melanogaster milt
, milt
, milt
Mislocalization of mitochondria Axonal transport of
Mitochondrial Rho-GTPase dMiro dMiro, Miro D. melanogaster B682, Sd10, Sd23, Sd26, Sd32 Mislocalization of mitochondria Axonal transport of
Apolipoprotein E ApoE ApoE M. musculus Overexpression Accumulation of mitochondria,
Syn, neurofilaments
NM_009696 NP_033826 [101]
Heatshock protein 27 HSPB1 HSPB1, hsp27 H. sapiens R127W, S135F, R136W,
T151I, P182L
Charcot-Marie-Tooth disease Molecular chaperone NM_001540 NP_001531 [102]
HSPB1 HSPB1, hsp27 Cell culture P182L Insoluble intracellular aggregates,
sequestration of p150, neurofilaments
Molecular chaperone NM_001540 NP_001531 [103]
Paraplegin Spg7 Paraplegin M. musculus knockout Axonal accumulations of organelles,
neurofilaments, Hereditary Spastic
Paraplegia SPG7
Metalloprotease NM_153176 NP_694816 [104]
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cytoplasmic dynein and dynactin in motor neurons, and
motor neuron degeneration [43,44]. Three additional lesions
in the p150
subunit of dynactin have also been identified
in patients with amyotrophic lateral sclerosis [45].
Motor proteins bind to transmembrane proteins on the
cargo surface directly, or indirectly, via intermediary scaffold
proteins (Figure 1) [6,46]. The cJun NH
-terminal kinase
(JNK) interacting protein (JIP) group is a class of proteins that
may link the kinesin motor to cargo and also act as a scaffold
for components of the stress-activated JNK kinase signaling
pathway [47]. This implies that the subcellular localization of
the JNK signaling complex in the neuron may be regulated by
vesicular axonal transport or conversely that kinesin motor
activity during axonal transport may itself be regulated via
the JNK signaling pathway. In support of the latter, deletion
of JNK and JNK kinase results in the mislocalization of
synaptic vesicle components in C. elegans [48], although this
could be due to a requirement of JNK to regulate
microtubule dynamics [49]. The JIP1 and JIP2 proteins are
thought to link kinesin with apolipoprotein E receptor 2
(ApoER2) on cargo [50,51]. Aplip1, the Drosophila JIP1
homolog, is required in axonal vesicle transport and,
curiously, the retrograde transport of mitochondria [52].
Sunday Driver (Syd)/JIP3 was identified in Drosophila as a
scaffold protein possibly required for the interaction of
kinesin with vesicles transported in the axon [53].
Interestingly, Syd/JIP3 is implicated as a transport-dependent
positive-injury signal in the response to axonal damage [54].
Another interesting process was recently found in studies
of the motor domain of KIF5 which has been suggested to
interpret variations in microtubule structure in the neuronal
cell body to ensure that cargo is directed into the axon [55].
The mechanism by which this occurs is unclear, but
microtubule-associated proteins on the surface of
microtubules are probable candidates. The predominant
microtubule-associated protein in the axon is tau, which
promotes microtubule assembly and stability. Mutations in
tau not only impair its ability to bind, stabilize, and assemble
microtubules [56,57], but also retard its slow transport in the
axon [58]. When tau is overexpressed [59,60] or abnormally
phosphorylated [61,62], it forms aggregates that may
physically block the fast anterograde transport of
mitochondria, neurofilaments, peroxisomes, and vesicles
carrying the amyloid precursor protein (APP). The
retrograde axonal transport of signaling endosomes that
provide neurotrophic support for the neuron may also be
blocked and prevented from reaching the cell body [63].
The Drosophila proteins Milton and mitochondrial GTPase
Miro are also required for the transport of mitochondria [64–
66]. Lesions in Milton and Miro result in the specific failure of
mitochondria to be transported anterogradely, and they
consequently accumulate in the cell body, although the
transport of synaptic vesicles is unaffected.
Links between Axonal Transport and Human
Neurodegenerative Disease
Defects in axonal transport have been indirectly linked to a
number of progressive human neurodegenerative diseases
including Alzheimer disease (AD), Huntington disease (HD),
and amyotrophic lateral sclerosis (ALS). One common feature
of these diseases is that the proteins encoded by genes linked
Table 3. Continued
Gene Common
Organism Lesion Phenotype/Disease Inferred
for mRNA
for Protein
Tau tau Tau M. musculus R406W Retarded axonal transport of tau Microtubule binding NM_016835
tau Tau Cell culture Overexpression htau40, tau23, K35 Retarded axonal transport of tau,
APP, mitochondria, vesicles,
Microtubule binding NM_016835
tau Tau M. musculus Overexpression htau40, T44 Axonal accumulation of mitochondria,
neurofilaments, vesicles
Microtubule binding NM_016835
Knockout or mutagenic lesions in the listed genes that encode for proteins other than motor components result in defective axonal transport. Accession numbers for each gene were obtained from the National Center for Biotechnology Information
(NCBI) database (, accessed 2 May 2006.
Denotes genes that have multiple accession numbers for different isoforms derived from alternative splicing.
SwissProt Database
DOI: 10.1371/journal.pgen.0020124.t003
PLoS Genetics | September 2006 | Volume 2 | Issue 9 | e1241281
to each disease are transported in the axon and can perturb
transport when manipulated; presenilin 1 and APP in AD, Cu/
Zn superoxide dismutase (SOD1) in ALS, and huntingtin (Htt)
in HD. Each disease is characterized by accumulations of
these or other proteins within axons, similar to defective
axonal transport phenotypes observed in animal models of
motor protein mutants.
The pathological hallmarks of AD include neurofibrillary
tangles of abnormally phosphorylated tau protein and
aggregates of amyloid- b (Ab) peptide resulting in neuritic
plaques in the brain [67]. The transmembrane protein APP,
the precursor of potentially neurotoxic Ab, is transported
anterogradely within vesicles in axons by the fast axonal
transport system [68]. Interestingly, APP may link the kinesin
motor either directly, or indirectly, via the JIP1 scaffold, to a
specific class of synaptic vesicles containing synapsin 1,
growth-associated protein 43 (GAP-43), along with b-
secretase and presenilin 1, two components responsible for
processing Ab from APP [69,70]. Deletion of the APP
homolog Appl in Drosophila results in defective axonal
transport including axonal accumulation phenotypes [71].
Overexpression of human APP causes similar phenotypes
that are enhanced by genetic reduction in kinesin and
suppressed by genetic reduction in cytoplasmic dynein [71].
These findings suggest that APP plays a central role in the
axonal transport of a specific class of vesicle and that
disruption in this transport, through lesions in APP or APP-
interacting components, may result in axonal blockages, a
possible causative factor in the development of AD.
HD is a progressive neurodegenerative disorder caused by
expansion of CAG triplet repeats in the coding sequence of
the huntingtin gene resulting in an expanded polyglutamine
tract (polyQ) in the Htt protein and a toxic gain of function.
Interestingly, both Htt and the Huntingtin-associated
protein 1 (HAP1) are anterogradely and retrogradely
transported in axons [72]. HAP1 interacts with the
anterograde motor kinesin via the Klc subunit and is thought
to interact with the retrograde motor cytoplasmic dynein
through an association with the p150
subunit of
dynactin [73–75]. Recent studies raise the possibility of a link
between axonal transport defects and the onset of HD. In
Drosophila, both a reduction of Htt protein and the
overexpression of proteins containing polyQ repeats result
in axonal transport defects [76]. Full-length mutant Htt also
impairs vesicular and mitochondrial transport in mouse
neurons [77]. Although the mechanism of axonal transport
disruption remains unclear, one possibility is that toxic Htt
titrates soluble motor protein components into axonal
aggregates that physically block transport. One class of
vesicle potentially affected are those containing brain-
derived neurotrophic factor which would result in loss of
neurotrophic support and neuronal toxicity [77,78].
Interestingly, in transport studies performed on extruded
squid axoplasm, recombinant Htt fragments with polyQ
expansions inhibited fast axonal transport in the absence of
aggregate formation [79]. This suggests that polyQ aggregates
may not be necessary for axonal transport disruption, but
may contribute to or enhance neuronal toxicity. Clearly, a
more comprehensive analysis is required to elucidate the
mechanism of polyQ toxicity.
Lesions in the ubiquitously expressed enzyme SOD1 are a
cause of rare hereditary ALS [80,81]. Mouse models of
hereditary ALS have been generated by transgenic expression
of mutant SOD1. These animals have impaired slow axonal
transport with axonal accumulations of neurofilaments and
tubulin [82–85]. Similarly, large axonal swellings with
neurofilament accumulations, consistent with a failure in
axonal transport, are observed in patients with ALS [86,87]. It
has been suggested that SOD1 may specifically inhibit
retrograde axonal transport [88]. The potential involvement
of cytoplasmic dynein in ALS was further highlighted by the
identification of a number of lesions in the motor binding
domain of dynactin subunit p150
in ALS patients [45].
Additional support comes from the observation that the
cytoplasmic dynein mutations Loa and Cra1 revert axonal
transport defects of ALS mice, attenuating motor neuron
degeneration resulting in delayed onset of disease and
extended lifespan [89,90].
Conclusions and Future Directions
Although a potential link between axonal transport
disorders and neurodegenerative disease has been suggested,
a number of critical questions remain unanswered. For
example, recent evidence indicates that axonal transport is
disrupted in mouse models of ALS, HD, and AD long before
detectable pathological hallmarks of the disease are observed
[77,83,91]. Similarly, comparable pathology may exist early in
these human diseases. Yet, it remains unclear whether these
changes are causes or consequences of the disease process.
Unraveling these issues will require a better understanding of
how axonal transport is controlled and which components
contribute to the various pathways. In several cases, it is not
known whether human mutations represent loss of function
or give rise to dominant negative effects, resulting in toxic
proteins that titrate or poison axonal transport components.
As a result, the effect on axonal transport could be specific
and cause the disruption of only a single class of transported
material, or nonspecific and reduce or physically block
multiple transport pathways through the aggregation of
transported cargoes into axonal blockages. It is likely that
both mechanisms occur, depending on the nature of the
lesion and the motor component involved. Finally, while
genetics in model systems will continue to clarify
mechanisms, further investigations of heritable neurological
disorders in humans may lead to the identification of
additional motor proteins or accessory components required
for axonal transport. In any event, a more comprehensive
understanding of axonal transport may lead to the
development of novel therapies for the treatment of
neurodegenerative disorders.
We apologize to those authors whose work was not cited due to space
limitations. The authors thank members of the Goldstein laboratory,
Sameer Shah, Carole Weaver, Kristina Schimmelpfeng, Tomas
Falzone, Shermali Gunawardena, and Louise Parker for thoughtful
discussions and critical reading of the manuscript. Jason Duncan
would like to thank Caitlin Foreman for her guidance and support
during the writing of this manuscript.
Author contributions. JED and LSBG wrote the paper.
Funding. LSBG is an investigator of the Howard Hughes Medical
Competing interests. The authors have declared that no competing
interests exist.
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PLoS Genetics | September 2006 | Volume 2 | Issue 9 | e1241284
... Kinesins are a large family of 45 proteins that form the molecular motors responsible for anterograde axonal transport directed by the (+) end of the microtubule [26]. The kinesin-1 motor consists of a kinesin heavy chain dimer, encoded by KIF5A, KIF5B and KIF5C, as well as a dimer of kinesin light chains [27]. Kinesin motors facilitate the anterograde transport of synaptic vesicles, mitochondria, ion channels, adhesion molecules and mRNA from the cell body toward synaptic terminals and into the growth cone [13,25]. ...
... For instance, the adaptor proteins Miro and Milton are necessary to anchor mitochondria to the anterograde motor kinesin-1 [28], whilst numerous adaptor proteins are thought to be involved in binding mitochondria to dynein for retrograde transport [4], although the precise mechanisms for mitochondrial tethering remain unclear. In retrograde transport, the function of dynein is dependent on the dynactin complex (includes proteins encoded by DCTN 1/2) as it links cytoplasmic dynein to its cargo and regulates dynein activity [25,27]. Additionally, the activity of kinases, phosphatases, Rab-GTPases and Ca 2+ concentrations also regulate the cargo-motor associations [14]. ...
... Kinesins are a large family of 45 proteins that form the molecular motors responsible for anterograde axonal transport directed by the (+) end of the microtubule [26]. The kinesin-1 motor consists of a kinesin heavy chain dimer, encoded by KIF5A, KIF5B and KIF5C, as well as a dimer of kinesin light chains [27]. Kinesin motors facilitate the anterograde transport of synaptic vesicles, mitochondria, ion channels, adhesion molecules and mRNA from the cell body toward synaptic terminals and into the growth cone [13,25]. ...
... For instance, the adaptor proteins Miro and Milton are necessary to anchor mitochondria to the anterograde motor kinesin-1 [28], whilst numerous adaptor proteins are thought to be involved in binding mitochondria to dynein for retrograde transport [4], although the precise mechanisms for mitochondrial tethering remain unclear. In retrograde transport, the function of dynein is dependent on the dynactin complex (includes proteins encoded by DCTN 1/2) as it links cytoplasmic dynein to its cargo and regulates dynein activity [25,27]. Additionally, the activity of kinases, phosphatases, Rab-GTPases and Ca 2+ concentrations also regulate the cargo-motor associations [14]. ...
Full-text available
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective, early degeneration of motor neurons in the brain and spinal cord. Motor neurons have long axonal projections, which rely on the integrity of neuronal cytoskeleton and mitochondria to regulate energy requirements for maintaining axonal stability, anterograde and retrograde transport, and signaling between neurons. The formation of protein aggregates which contain cytoskeletal proteins, and mitochondrial dysfunction both have devastating effects on the function of neurons and are shared pathological features across several neurodegenerative conditions, including ALS, Alzheimer's disease, Parkinson's disease, Huntington’s disease and Charcot-Marie-Tooth disease. Furthermore, it is becoming increasingly clear that cytoskeletal integrity and mitochondrial function are intricately linked. Therefore, dysregulations of the cytoskeletal network and mitochondrial homeostasis and localization, may be common pathways in the initial steps of neurodegeneration. Here we review and discuss known contributors, including variants in genetic loci and aberrant protein activities, which modify cytoskeletal integrity, axonal transport and mitochondrial localization in ALS and have overlapping features with other neurodegenerative diseases. Additionally, we explore some emerging pathways that may contribute to this disruption in ALS.
... Predominantly targeting the elderly as the most frequent culprit for dementia [41], Alzheimer's disease (AD) is believed to tightly accompany ageing with varying symptoms from slight memory loss in early stages to profound disability and even fatality in acute stages [42][43][44]. Further studies on the pathogenesis of the disease have disclosed that abnormally functioning proteins are widely accepted to be the leading causative agents associated with axonal swelling and neuronal dysfunction, aggregation of which culminates with the disruption of neural communication and signalling, particularly in the cortical and limbic areas [45,46]. Meticulously, empirical evidence suggests the principal hallmarks of this proteinopathy include (I) amyloid-β (Aβ) dense-core neuritic plaques (NPs; senile plaques) and diffuse plaques (DPs) derived from sequentially β-and γ-secretaseproteolysed transmembrane protein amyloid precursor protein (APP) together with (II) hyperphosphorylated τ protein (P-τ) constituting neurofibrillary tangles (NFTs) [47,48], which assemble beyond and within the plasma membrane, respectively. ...
Numerous factors are implicated in the onset and progression of ageing and neurodegenerative disorders, with defects in cell energy supply and free radicals regulation designated as being the main functions of mitochondria and highly accentuated in plentiful studies. Hence, analysing the role of mitochondria as one of the main factors implicated in these disorders could undoubtedly come in handy with respect to disease prevention and treatment. In this review, first, we will explore how mitochondria account for neurodegenerative disorders and ageing and later will draw the various pathways contributing to mitochondrial dysfunction in their distinct way. Also, we will discuss the deviation-countering mechanisms, particularly mitophagy, a subset of autophagy known as a much larger cellular defence mechanism and regulatory system, along with its potential therapeutic effects. Last but not least, we will be highlighting the mitochondrial transfer experiments with animal models of neurodegenerative disorders.
... This motor protein is only responsible for movement along the microtubule and cannot bind to cargo. Another protein called dynactin can bind to the cargo (Duncan & Goldstein, 2006). Then dynactin binds to dynein and transports the dynein protein and cargo like a tow truck (Fig. 8). ...
Introduction Like other cells in the body, nerve cells need many proteins and substances to maintain homeostasis. As we know, the transcription and translation of proteins and necessary cellular substances occurs in the cell nucleus. The nucleus of nerve cell is located in the cell body. Another part of the nerve cell is "Axon", which has a long structure. Even in some nerve cells axon's length reaches up to 1000 mm. On the other hand, all parts of the neuron need substances and proteins synthesized in nucleus locating in the cell body. Therefore, a mechanism is necessary to express the movement of materials from nucleus along the axon. The movement of materials along the axon is called 'Axoplasmic Transport'. It seems that disturbances in axoplasmic transport can cause various neuronal problems. The purpose of this study is to investigate the mechanism of axoplasmic transport and its types; moreover, the possible effect of exercise on this transition will be discussed.
... Dynactin 1, which binds to microtubules, is a motor protein responsible for the retrograde transport of various proteins and vesicles [270]. ALS and slowly progressing, autosomal dominant, distal hereditary motor neuropathy in vocal paresis are due to loss-of-function mutations in dynactin 1 [271][272][273]. ...
Full-text available
Amyotrophic lateral sclerosis (ALS) is an intractable disease that causes respiratory failure leading to mortality. The main locus of ALS is motor neurons. The success of antisense oligonucleotide (ASO) therapy in spinal muscular atrophy (SMA), a motor neuron disease, has triggered a paradigm shift in developing ALS therapies. The causative genes of ALS and disease-modifying genes, including those of sporadic ALS, have been identified one after another. Thus, the freedom of target choice for gene therapy has expanded by ASO strategy, leading to new avenues for therapeutic development. Tofersen for superoxide dismutase 1 (SOD1) was a pioneer in developing ASO for ALS. Improving protocols and devising early interventions for the disease are vital. In this review, we updated the knowledge of causative genes in ALS. We summarized the genetic mutations identified in familial ALS and their clinical features, focusing on SOD1, fused in sarcoma (FUS), and transacting response DNA-binding protein. The frequency of the C9ORF72 mutation is low in Japan, unlike in Europe and the United States, while SOD1 and FUS are more common, indicating that the target mutations for gene therapy vary by ethnicity. A genome-wide association study has revealed disease-modifying genes, which could be the novel target of gene therapy. The current status and prospects of gene therapy development were discussed, including ethical issues. Furthermore, we discussed the potential of axonal pathology as new therapeutic targets of ALS from the perspective of early intervention, including intra-axonal transcription factors, neuromuscular junction disconnection, dysregulated local translation, abnormal protein degradation, mitochondrial pathology, impaired axonal transport, aberrant cytoskeleton, and axon branching. We simultaneously discuss important pathological states of cell bodies: persistent stress granules, disrupted nucleocytoplasmic transport, and cryptic splicing. The development of gene therapy based on the elucidation of disease-modifying genes and early intervention in molecular pathology is expected to become an important therapeutic strategy in ALS.
... Scaffold proteins can act as adaptors and also bind to members of the mitogen-activated protein family of kinases (MAPKs) (Dhanasekaran et al., 2007). For example, the MAPK-associated scaffold protein, JIP3/Sunday Driver, binds to kinesin-1 and has an evolutionarily conserved role in the axonal transport of synapsedirected vesicles (Cockburn et al., 2018;Duncan and Goldstein, 2006). Similarly, the scaffold protein JIP1 can bind to kinesin-1 (Fu and Holzbaur, 2014), and also to the GluA4 AMPAR subunit (Vieira et al., 2010); however, a role for JIP1 in AMPAR transport has not been established. ...
Synaptic plasticity depends on rapid experience-dependent changes in the number of neurotransmitter receptors. Previously, we demonstrated that motor-mediated transport of AMPA receptors (AMPARs) to and from synapses is a critical determinant of synaptic strength. Here, we describe two convergent signaling pathways that coordinate the loading of synaptic AMPARs onto scaffolds, and scaffolds onto motors, thus providing a mechanism for experience-dependent changes in synaptic strength. We find that an evolutionarily conserved JIP-protein scaffold complex and two classes of mitogen-activated protein kinase (MAPK) proteins mediate AMPAR transport by kinesin-1 motors. Genetic analysis combined with in vivo, real-time imaging in Caenorhabditis elegans revealed that CaMKII is required for loading AMPARs onto the scaffold, and MAPK signaling is required for loading the scaffold complex onto motors. Our data support a model where CaMKII signaling and a MAPK-signaling pathway cooperate to facilitate the rapid exchange of AMPARs required for early stages of synaptic plasticity.
Axons are long slender cylindrical projections of neurons that enable these cells to communicate directly with other cells in the body over long distances, up to a meter or more in large animals. Remarkably, however, most axonal components originate in the nerve cell body, at one end of the axon, and must be shipped out along the axon by mechanisms of intracellular motility. In addition, signals from the axon and its environment must be conveyed back to the nerve cell body to modulate the nature and composition of the outbound traffic. The outward movement from the cell body toward the axon tip is called anterograde transport and the movement in the opposite direction, back toward the cell body, is called retrograde transport. This bidirectional transport, known collectively as axonal transport, is not fundamentally different from the pathways of macromolecular and membrane traffic found in other parts of the neuron, or indeed in any eukaryotic cell, but it is unique for the volume and scale of the traffic required to maintain these long processes.
Neurodegenerative diseases (NDs) are heterogeneous neurological disorders characterized by a progressive loss of selected neuronal populations. A significant risk factor for most NDs is aging. Considering the constant increase in life expectancy, NDs represent a global public health burden. Axonal transport (AT) is a central cellular process underlying the generation and maintenance of neuronal architecture and connectivity. Deficits in AT appear to be a common thread for most, if not all, NDs. Neuroinflammation has been notoriously difficult to define in relation to NDs. Inflammation is a complex multifactorial process in the CNS, which varies depending on the disease stage. Several lines of evidence suggest that AT defect, axonopathy and neuroinflammation are tightly interlaced. However, whether these impairments play a causative role in NDs or are merely a downstream effect of neuronal degeneration remains unsettled. We still lack reliable information on the temporal relationship between these pathogenic mechanisms, although several findings suggest that they may occur early during ND pathophysiology. This article will review the latest evidence emerging on whether the interplay between AT perturbations and some aspects of CNS inflammation can participate in ND etiology, analyze their potential as therapeutic targets, and the urge to identify early surrogate biomarkers.
In this study a manganese-enhanced magnetic resonance imaging (MEMRI) method was developed for mice for measuring axonal transport (AXT) rates in real time in olfactory receptor neurons, which project from the olfactory epithelium to the olfactory neuronal layer of the olfactory bulb. Using this MEMRI method, two major experiments were conducted: 1) an evaluation of the effects of age on AXT rates and 2) an evaluation of the brain-penetrant, microtubule-stabilizing agent, Epothilone D for effect on AXT rates in aged mice. In these studies, we improved upon previous MEMRI approaches to develop a method where real-time measurements (32 time points) of AXT rates in mice can be determined over a single (approximately 100 min) scanning session. In the age comparisons, AXT rates were significantly higher in young (mean age ∼4.0 months old) versus aged (mean age ∼24.5 months old) mice. Moreover, in aged mice, eight weeks of treatment with Epothilone D, (0.3 and 1.0 mg/kg) was associated with statistically significant increases in AXT rates compared to vehicle-treated subjects. These experiments conducted in a living mammalian model (i.e., wild type, C57BL/6 mice), using a new modified MEMRI method, thus provide further evidence that the process of aging leads to decreases in AXT rates in the brain and they further support the argument that microtubule-based therapeutic strategies designed to improve AXT rates have potential for age-related neurological disorders.
The use of primary neuronal cultures generated from Drosophila tissue provides a powerful model for studies of transport mechanisms. Cultured fly neurons provide similarly detailed subcellular resolution and applicability of pharmacology or fluorescent dyes as mammalian primary neurons. As an experimental advantage for the mechanistic dissection of transport, fly primary neurons can be combined with the fast and highly efficient combinatorial genetics of Drosophila , and genetic tools for the manipulation of virtually every fly gene are readily available. This strategy can be performed in parallel to in vivo transport studies to address relevance of any findings. Here we will describe the generation of primary neuronal cultures from Drosophila embryos and larvae, the use of external fluorescent dyes and genetic tools to label cargo, and the key strategies for live imaging and subsequent analysis.
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KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport.
Loss of synapses and dying back of axons are considered early events in brain degeneration during Alzheimer’s disease. This is accompanied by an aberrant behavior of the microtubule-associated protein tau (hyperphosphorylation, aggregation). Since microtubules are the tracks for axonal transport, we are testing the hypothesis that tau plays a role in the malfunctioning of transport. Experiments with various neuronal and non-neuronal cells show that tau is capable of reducing net anterograde transport of vesicles and cell organelles by blocking the microtubule tracks. Thus, a misregulation of tau could cause the starvation of synapses and enhanced oxidative stress, long before tau detaches from microtubules and aggregates into Alzheimer neurofibrillary tangles. In particular, the transport of amyloid precursor protein is retarded when tau is elevated, suggesting a possible link between the two key proteins that show abnormal behavior in Alzheimer’s disease.
Cytoplasmic dynein, a minus end–directed, microtubule-based motor protein, is thought to drive the movement of membranous organelles and chromosomes. It is a massive complex that consists of multiple polypeptides. Among these polypeptides, the cytoplasmic dynein heavy chain (cDHC) constitutes the major part of this complex. To elucidate the function of cytoplasmic dynein, we have produced mice lacking cDHC by gene targeting. cDHC−/− embryos were indistinguishable from cDHC+/−or cDHC+/+ littermates at the blastocyst stage. However, no cDHC−/− embryos were found at 8.5 d postcoitum. When cDHC−/− blastocysts were cultured in vitro, they showed interesting phenotypes. First, the Golgi complex became highly vesiculated and distributed throughout the cytoplasm. Second, endosomes and lysosomes were not concentrated near the nucleus but were distributed evenly throughout the cytoplasm. Interestingly, the Golgi “fragments” and lysosomes were still found to be attached to microtubules. These results show that cDHC is essential for the formation and positioning of the Golgi complex. Moreover, cDHC is required for cell proliferation and proper distribution of endosomes and lysosomes. However, molecules other than cDHC might mediate attachment of the Golgi complex and endosomes/lysosomes to microtubules.
AMYOTROPHIC lateral sclerosis (ALS) is a degenerative disorder of motor neurons in the cortex, brainstem and spinal cord1,2. Its cause is unknown and it is uniformly fatal, typically within five years3. About 10% of cases are inherited as an autosomal dominant trait, with high penetrance after the sixth decade4,5. In most instances, sporadic and autosomal dominant familial ALS (FALS) are clinically similar4,6,7. We have previously shown that in some but not all FALS pedigrees the disease is linked to a genetic defect on chromosome 21q (refs 8, 9). Here we report tight genetic linkage between FALS and a gene that encodes a cytosolic, Cu/Zn-binding superoxide dismutase (SOD1), a homodimeric metalloenzyme that catalyzes the dismutation of the toxic superoxide anion O2.- to O2 and H2O2 (ref. 10). Given this linkage and the potential role of free radical toxicity in other neurodenegerative disorders11, we investigated SOD1 as a candidate gene in FALS. We identified 11 different SOD1 missense mutations in 13 different FALS families.
Although cytoplasmic dynein is known to attach to microtubules and translocate toward their minus ends, dynein's ability to serve in vitro as a minus end-directed transporter of membranous organelles depends on additional soluble factors. We show here that a approximately 20S polypeptide complex (referred to as Activator I; Schroer, T. A., and M.P. Sheetz. 1991a. J. Cell Biol. 115:1309-1318.) stimulates dynein-mediated vesicle transport. A major component of the activator complex is a doublet of 150-kD polypeptides for which we propose the name dynactin (for dynein activator). The 20S dynactin complex is required for in vitro vesicle motility since depletion of it with a mAb to dynactin eliminates vesicle movement. Cloning of a brain specific isoform of dynactin from chicken reveals a 1,053 amino acid polypeptide composed of two coiled-coil alpha-helical domains interrupted by a spacer. Both this structural motif and the underlying primary sequence are highly conserved in vertebrates with 85% sequence identity within a central 1,000-residue domain of the chicken and rat proteins. As abundant as dynein, dynactin is ubiquitously expressed and appears to be encoded by a single gene that yields at least three alternative isoforms. The probable homologue in Drosophila is the gene Glued, whose protein product shares 50% sequence identity with vertebrate dynactin and whose function is essential for viability of most (and perhaps all) cells in the organism.
We cloned a new member of the murine brain kinesin superfamily, KIF3B, and found that its amino acid sequence is highly homologous but not identical to KIF3A, which we previously cloned and named KIF3 (47% identical). KIF3B is localized in various organ tissues and developing neurons of mice and accumulates with anterogradely moving membranous organelles after ligation of nerve axons. Immunoprecipitation assay of the brain revealed that KIF3B forms a complex with KIF3A and three other high molecular weight (approximately 100 kD)-associated polypeptides, called the kinesin superfamily-associated protein 3 (KAP3). In vitro reconstruction using baculovirus expression systems showed that KIF3A and KIF3B directly bind with each other in the absence of KAP3. The recombinant KIF3A/B complex (approximately 50-nm rod with two globular heads and a single globular tail) demonstrated plus end-directed microtubule sliding activity in vitro. In addition, we showed that KIF3B itself has motor activity in vitro, by making a complex of wild-type KIF3B and a chimeric motor protein (KIF3B head and KIF3A rod tail). Subcellular fractionation of mouse brain homogenates showed a considerable amount of the native KIF3 complex to be associated with membrane fractions other than synaptic vesicles. Immunoprecipitation by anti-KIF3B antibody-conjugated beads and its electron microscopic study also revealed that KIF3 is associated with membranous organelles. Moreover, we found that the composition of KAP3 is different in the brain and testis. Our findings suggest that KIF3B forms a heterodimer with KIF3A and functions as a new microtubule-based anterograde translocator for membranous organelles, and that KAP3 may determine functional diversity of the KIF3 complex in various kinds of cells in vivo.
Kinesin superfamily proteins (KIFs) comprise several dozen molecular motor proteins. The KIF3 heterotrimer complex is one of the most abundantly and ubiquitously expressed KIFs in mammalian cells. To unveil the functions of KIF3, microinjection of function-blocking monovalent antibodies against KIF3 into cultured superior cervical ganglion (SCG) neurons was carried out. They significantly blocked fast axonal transport and brought about inhibition of neurite extension. A yeast two-hybrid binding assay revealed the association of fodrin with the KIF3 motor through KAP3. This was further confirmed by using vesicles collected from large bundles of axons (cauda equina), from which membranous vesicles could be prepared in pure preparations. Both immunoprecipitation and immunoelectron microscopy indicated the colocalization of fodrin and KIF3 on the same vesicles, the results reinforcing the evidence that the cargo of the KIF3 motor consists of fodrin-associating vesicles. In addition, pulse-labeling study implied partial comigration of both molecules as fast flow components. Taken together, the KIF3 motor is engaged in fast axonal transport that conveys membranous components important for neurite extension.
Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97–amino acid polypeptide that is 57% identical (70% similar) to the 105–amino acid Chlamydomonas outer arm dynein–associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.