Andrew J. Grierson

Role of Axonal Transport in Neurodegenerative Diseases

Many major human neurodegenerative diseases, including Alzheimers disease, Parkinsons disease, and amyotrophic lateral sclerosis (ALS), display axonal pathologies including abnormal accumulations of proteins and organelles. Such pathologies highlight damage to the axon as part of the pathogenic process and, in particular, damage to transport of cargoes through axons. Indeed, we now know that disruption of axonal transport is an early and perhaps causative event in many of these diseases. Here, we review the role of axonal transport in neurodegenerative disease.

Molecular pathways of motor neuron injury in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a genetically diverse disease. At least 15 ALS-associated gene loci have so far been identified, and the causative gene is known in approximately 30% of familial ALS cases. Less is known about the factors underlying the sporadic form of the disease. The molecular mechanisms of motor neuron degeneration are best understood in the subtype of disease caused by mutations in superoxide dismutase 1, with a current consensus that motor neuron injury is caused by a complex interplay between multiple pathogenic processes. A key recent finding is that mutated TAR DNA-binding protein 43 is a major constituent of the ubiquitinated protein inclusions in ALS, providing a possible link between the genetic mutation and the cellular pathology. New insights have also indicated the importance of dysregulated glial cell–motor neuron crosstalk, and have highlighted the vulnerability of the distal axonal compartment early in the disease course. In addition, recent studies have suggested that disordered RNA processing is likely to represent a major contributing factor to motor neuron disease. Ongoing research on the cellular pathways highlighted in this Review is predicted to open the door to new therapeutic interventions to slow disease progression in ALS.

Familial amyotrophic lateral sclerosis-linked SOD1 mutants perturb fast axonal transport to reduce axonal mitochondria content

Amyotrophic lateral sclerosis (ALS) is a late-onset neurological disorder characterized by death of motoneurons. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) cause familial ALS but the mechanisms whereby they induce disease are not fully understood. Here, we use time-lapse microscopy to monitor for the first time the effect of mutant SOD1 on fast axonal transport (FAT) of bona fide cargoes in living neurons. We analyzed FAT of mitochondria that are a known target for damage by mutant SOD1 and also of membrane-bound organelles (MBOs) using EGFP-tagged amyloid precursor protein as a marker. We studied FAT in motor neurons derived from SOD1G93A transgenic mice that are a model of ALS and also in cortical neurons transfected with SOD1G93A and three further ALS-associated SOD1 mutants. We find that mutant SOD1 damages transport of both mitochondria and MBOs, and that the precise details of this damage are cargo-specific. Thus, mutant SOD1 reduces transport of MBOs in both anterograde and retrograde directions, whereas mitochondrial transport is selectively reduced in the anterograde direction. Analyses of the characteristics of mitochondrial FAT revealed that reduced anterograde movement involved defects in anterograde motor function. The selective inhibition of anterograde mitochondrial FAT enhanced their net retrograde movement to deplete mitochondria in axons. Mitochondria in mutant SOD1 expressing cells also displayed features of damage. Together, such changes to mitochondrial function and distribution are likely to compromise axonal function. These alterations represent some of the earliest pathological features so far reported in neurons of mutant SOD1 transgenic mice.

Mitochondrial function and actin regulate dynamin-related protein 1-dependent mitochondrial fission.

Mitochondria display a variety of shapes, ranging from small and spherical or the classical tubular shape to extended networks. Shape transitions occur frequently and include fusion, fission, and branching. It was reported that some mitochondrial shape transitions are developmentally regulated, whereas others were linked to disease or apoptosis. However, if and how mitochondrial function controls mitochondrial shape through regulation of mitochondrial fission and fusion is unclear. Here, we show that inhibitors of electron transport, ATP synthase, or the permeability transition pore (mtPTP) induced reversible mitochondrial fission. Mitochondrial fission depended on dynamin-related protein 1 (DRP1) and F-actin: Disruption of F-actin attenuated fission and recruitment of DRP1 to mitochondria. In contrast, uncoupling of electron transport and oxidative phosphorylation caused mitochondria to adopt a distinct disk shape. This shape change was independent of the cytoskeleton and DRP1 and was most likely caused by swelling. Thus, disruption of mitochondrial function rapidly and reversibly altered mitochondrial shape either by activation of DRP1-dependent fission or by swelling, indicating a close relationship between mitochondrial fission, shape, and function. Furthermore, our results suggest that the actin cytoskeleton is involved in mitochondrial fission by facilitating mitochondrial recruitment of DRP1.

Systemic Delivery of scAAV9 Expressing SMN Prolongs Survival in a Model of Spinal Muscular Atrophy

An adeno-associated virus vector expressing the survival motor neuron protein rescues mice with spinal muscular atrophy. Enough Protein to Reverse Spinal Muscular Atrophy A common neuromuscular disease, spinal muscular atrophy (SMA) causes ever-worsening muscle weakness, usually in babies or young children, almost always resulting in early death. The culprit is a defective gene—survival motor neuron (SMN)—that must be inherited from both parents for the child to be affected. A second SMN gene is usually incorrectly spliced and so is nonfunctional. To treat this disease, researchers have set their sights on delivering a replacement SMN to the motor neurons. The hope has been that gene therapy methods could be used to generate enough normal SMN protein to restore neuronal innervation of muscles in the affected children. Valori et al. have now improved on previous attempts to implement such a treatment in mice with an SMN-like disease. Their new gene therapy vector makes enough functional SMN protein to improve the agility of the affected animals and markedly increase their survival. To find gene therapy methods for SMA that work in animals before trying these treatments in humans, researchers had created mice with the disease. The mouse SMN gene was deleted (mice have only one copy) and replaced with the two human genes—the defective disease-causing form and its poorly spliced sibling. Valori et al. then treated these animals with a gene therapy vector that they had tested in fibroblasts. They coupled a fast, efficient virus (a modified self-complementary adeno-associated virus) to the coding sequence for the SMN protein, optimized for efficient codon usage. A single injection of this vector to new born mice with SMA improved their ability to move and perform physical tests. These mice, which usually die at 2 weeks of age, also showed markedly increased survival times. Subsequent immunohistochemistry showed that the introduced gene was expressed all over the body of the animals but particularly in the lumbar spinal cord, muscle, and liver. These results bring us one step closer to a successful gene therapy treatment for patients with SMA. The vector used here produces large amounts of replacement SMN protein, a goal that had not been achieved with previous approaches. Another noteworthy feature of this vector is that it increases SMN expression in numerous cell types, not just motor neurons, which may be a clue that the critical defects in SMA may be located in more than one kind of cell. Spinal muscular atrophy is one of the most common genetic causes of death in childhood, and there is currently no effective treatment. The disease is caused by mutations in the survival motor neuron gene. Gene therapy aimed at restoring the protein encoded by this gene is a rational therapeutic approach to ameliorate the disease phenotype. We previously reported that intramuscular delivery of a lentiviral vector expressing survival motor neuron increased the life expectancy of transgenic mice with spinal muscular atrophy. The marginal efficacy of this therapeutic approach, however, prompted us to explore different strategies for gene therapy delivery to motor neurons to achieve a more clinically relevant effect. Here, we report that a single injection of self-complementary adeno-associated virus serotype 9 expressing green fluorescent protein or of a codon-optimized version of the survival motor neuron protein into the facial vein 1 day after birth in mice carrying a defective survival motor neuron gene led to widespread gene transfer. Furthermore, this gene therapy resulted in a substantial extension of life span in these animals. These data demonstrate a significant increase in survival in a mouse model of spinal muscular atrophy and provide evidence for effective therapy.

Neurofilament heavy chain side arm phosphorylation regulates axonal transport of neurofilaments

Neurofilaments possess side arms that comprise the carboxy-terminal domains of neurofilament middle and heavy chains (NFM and NFH); that of NFH is heavily phosphorylated in axons. Here, we demonstrate that phosphorylation of NFH side arms is a mechanism for regulating transport of neurofilaments through axons. Mutants in which known NFH phosphorylation sites were mutated to preclude phosphorylation or mimic permanent phosphorylation display altered rates of transport in a bulk transport assay. Similarly, application of roscovitine, an inhibitor of the NFH side arm kinase Cdk5/p35, accelerates neurofilament transport. Analyses of neurofilament movement in transfected living neurons demonstrated that a mutant mimicking permanent phosphorylation spent a higher proportion of time pausing than one that could not be phosphorylated. Thus, phosphorylation of NFH slows neurofilament transport, and this is due to increased pausing in neurofilament movement.

Mutations in CHMP2B in Lower Motor Neuron Predominant Amyotrophic Lateral Sclerosis (ALS)

Background Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3–10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis. Methodology/Principal Findings Sequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p.Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B. Conclusions/Significance We conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS. We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype.

Parkinson's disease alpha-synuclein mutations exhibit defective axonal transport in cultured neurons

α-Synuclein is a major protein constituent of Lewy bodies and mutations in α-synuclein cause familial autosomal dominant Parkinsons disease. One explanation for the formation of perikaryal and neuritic aggregates of α-synuclein, which is a presynaptic protein, is that the mutations disrupt α-synuclein transport and lead to its proximal accumulation. We found that mutant forms of α-synuclein, either associated with Parkinsons disease (A30P or A53T) or mimicking defined serine, but not tyrosine, phosphorylation states exhibit reduced axonal transport following transfection into cultured neurons. Furthermore, transfection of A30P, but not wild-type, α-synuclein results in accumulation of the protein proximal to the cell body. We propose that the reduced axonal transport exhibited by the Parkinsons disease-associated α-synuclein mutants examined in this study might contribute to perikaryal accumulation of α-synuclein and hence Lewy body formation and neuritic abnormalities in diseased brain.

Phosphorylation of thr668 in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3)

Threonine668 (thr668) within the carboxy‐terminus of the Alzheimers disease amyloid precursor protein (APP) is a known in vivo phosphorylation site. Phosphorylation of APPthr668 is believed to regulate APP function and metabolism. Thr668 precedes a proline, which suggests that it is targeted for phosphorylation by proline‐directed kinase(s). We have investigated the ability of four major neuronally active proline‐directed kinases, cyclin dependent protein kinase‐5, glycogen synthase kinase‐3β, p42 mitogen‐activated protein kinase and stress‐activated protein kinase‐1b, to phosphorylate APPthr668 and report here that SAPK1b induces robust phosphorylation of this site both in vitro and in vivo. This finding provides a molecular framework to link cellular stresses with APP metabolism in both normal and disease states.

Hereditary spastic paraparesis: disrupted intracellular transport associated with spastin mutation.

The commonest cause of hereditary spastic paraplegia (HSP) is mutation in the spastin gene. Both the normal function of spastin in the central nervous system and the mechanism by which mutation in spastin causes axonal degeneration are unknown. One hypothesis is that mutant spastin disrupts microtubule dynamics, causing an impairment of organelle transport on the microtubule network, which leads to degeneration in the distal parts of long axons. To study this neuronal and non‐neuronal cells were transfected with either wild type or mutant spastin proteins. We demonstrated evidence of a transient interaction of wild‐type spastin with microtubules, with resulting disassembly of microtubules, supporting a role for wild‐type spastin as a microtubule‐severing protein. Mutant spastin demonstrated an abnormal interaction with microtubules, colocalizing with but no longer severing microtubules. The abnormal interaction of mutant spastin with microtubules was demonstrated to be associated with an abnormal perinuclear clustering of mitochondria and peroxisomes, suggestive of an impairment of kinesin‐mediated intracellular transport. Our findings indicate that an abnormal interaction of mutant spastin with microtubules, which disrupts organelle transport on the microtubule cytoskeleton, is likely to be the primary disease mechanism in HSP caused by missense mutations in the spastin gene.