Molecular And Cellular Defects Leading To Neurodegenerative Diseases Biology Essay


Neurodegenerative diseases, which will become increasingly prevalent in our ageing society, share the common pathological feature of the accumulation of misfolded proteins and the subsequent formation of insoluble aggregates. Amongst the various pathogenic mechanisms proposed to cause these diseases, impaired axonal transport has been directly linked to neurodegeneration as defects in essential components of the axonal transport system, such as the kinesin and dynein microtubule-associated molecular motors and important components of the neuronal cytoskeleton, have been shown to cause various forms of neurodegenerative disease. Defective axonal transport may also be involved in the accumulation of aggregates, although it's role in the context of cause and effect has not been established. Defects in axonal transport therefore represent promising targets for the treatment and prevention of neurodegenerative diseases.

Keywords: axonal transport; retrograde and anterograde transport; molecular motor; kinesin; dynein; neuronal cytoskeleton; microtubule


Neurodegenerative diseases currently affect millions of people worldwide with the most prevalent of these disorders, Alzheimer's disease (AD) alone, affecting over 18 million people (Vas et al. 2002). The number of people affected by neurodegenerative diseases is set to increase significantly in our ageing populations as the risk of developing the majority of neurodegenerative diseases increases significantly with advancing age (World Health Organisation, Second United Nations Assembly on Ageing 2002, Active Ageing: a policy framework, It has been estimated that dementia affects over 24 million people and that the number affected will double every 20 years, reaching approximately 81 million by 2040 (Ferri et al. 2005).

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As further advances in medicine develop and average life expectancy continues to increase, there will be an increasing prevalence of neurodegenerative diseases in our society along with the significant emotional and financial strains that these diseases inevitably have on society and healthcare systems. Success in finding treatment and prevention strategies for neurodegenerative diseases is therefore becoming increasingly necessary.

Despite significant progress in the understanding of neurodegenerative diseases over the past several decades, due mainly to advances in the understanding of the genetic basis of these diseases, the molecular and cellular mechanisms are still far from being fully understood. Gaining a full understanding of the pathogenesis of these diseases is complicated as mechanisms may be interrelated in a complex viscous cycle, making it difficult to determine whether a specific defect is the cause or result of a disease.

It is vital that the molecular and cellular defects which affect the overall functioning of the nervous system, such as defects affecting integral components i.e. the 'rails' and 'motors' of the essential axonal transport system (the focus of this review), are identified and their interactions fully understood as these advances in our understanding are likely to provide potential targets for the successful treatment and prevention of these complex diseases.

Pathology of neurodegenerative diseases

There are a vast number of neurodegenerative diseases, the most prevalent and widely studied of which are Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). AD is the most common neurodegenerative disease and form of dementia worldwide, characterised clinically by progressive memory loss and cognitive dysfunction (McKhann et al. 1984). The regions of the brain most affected by neuron degeneration are the cortex, hippocampus and basal forebrain (Ross & Poirier 2004). The major histopathological features of AD are neuritic plaques, which consist mainly of the extra-cellular β-amyloid peptide (Aβ), a cleavage product of the amyloid precursor protein (APP), and neurofibrillary tangles (NFT), which mainly consist of the hyperphosphorylated form of the microtubule-associated protein tau (Reiman & Caselli 1999).

PD is the most predominant neurodegenerative movement disorder characterised by resting tremor, muscular rigidity, slow movements and postural instability (Jankovic 2008), caused by the degeneration of neurons in the substantia nigra, specifically the degeneration of dopaminergic neurons, and of neurons in the brain stem (Forno 1996). The major histopathological feature of PD are inclusion bodies known as Lewy bodies, of fibrillar, misfolded proteins found in the cytoplasm of neurons (Bossy-Wetzel et al. 2004). Lewy bodies consist mainly of aggregated α-synuclein protein and these aggregates are also found in neurites, thus termed Lewy neurites (Ross & Poirier 2004).

HD is an autosomal dominant inherited disease characterised by chorea and changes in personality and cognition (Charrin et al. 2005). HD is caused by the insertion of multiple CAG repeats in the huntingtin gene which codes for, and thereby causes an expansion of polyglutamine (polyQ) in the N-terminal of the huntingtin protein (Htt) which ultimately results in the selective loss of neurons in the striatum and cerebral cortex (Bossy-Wetzel et al. 2004). The mutated huntingtin gene causes the formation of nuclear and cytoplasmic inclusions in the regions of the brain where degeneration occurs (Forman et al. 2004).

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Due to the complexity of neurodegenerative diseases it seems unlikely that they are due to a single specific defect or mechanism of pathogenesis and more likely that they are due to a combination of cellular and molecular defects and interrelated events which are influenced by a variety of factors.

Protein aggregation in neurodegenerative diseases

Protein misfolding and the subsequent formation and progressive accumulation of insoluble aggregates are a common pathological feature of the majority of neurodegenerative diseases, including the described diseases most problematic in our society, and the presence of protein aggregates in a diverse range of these diseases strongly suggests that the accumulation of misfolded toxic proteins has a significant link to neurodegeneration. The misfolding of proteins specific to different neurodegenerative diseases, such as the β-amyloid peptide and microtubule-associated tau protein in AD, α-synuclein protein in PD and huntingtin protein in HD, is thought to "expose hydrophobic residues and unstructured chain segments that are normally concealed in the central parts of the proteins" (Gibbs & Braun 2008) which could explain why these misfolded proteins are highly prone to form insoluble aggregates. There are various theories regarding the mechanisms and extent of the involvement of protein aggregates in the pathogenesis of neurodegenerative diseases. It has been suggested that these protein aggregates may be the direct cause of neurodegenerative diseases through various mechanisms of toxicity and neurotoxic effects of intermediates at various stages in aggregate formation (Selkoe 2003; Forman et al. 2004; Jellinger 2009), or a result of other mechanisms which occur earlier and have a more direct role in the pathogenesis of neurodegenerative diseases (Ross & Poirier 2004; Gispert-Sanchez & Auburger 2006), or an inherent mechanism of protection against the degeneration of neurons (Gispert-Sanchez & Auburger 2006). The precise role of protein aggregates in the pathogenesis of neurodegenerative diseases is still to be established, although it seems highly likely that they have a central role in neurodegenerative diseases.

In the various neurodegenerative diseases, the misfolded proteins associated with each disease and their subsequent aggregates accumulate extracellulary and/or intracellularly in different regions of the brain which causes varying symptoms specific to the different diseases. In AD β-amyloid aggregates accumulate both intracellularly in the neuronal endoplasmic reticulum and extracellularly, in PD α-synuclein aggregates accumulate in neuronal cell bodies, axons and synapses and in HD the accumulation of aggregates occurs in the neuronal nuclei and cytoplasm (Hashimoto et al. 2003). The presence of aggregates in locations such as the cytosol, in which many important processes and reactions occur, which can be significantly affected by very minor changes in the surrounding conditions, suggests that there is a very high likelihood that their presence would affect important functions and processes in the neurons, which is likely to have negative effects and potentially lead to neurodegeneration.

Cells have adapted various mechanisms to eliminate, or at least minimise the accumulation of misfolded proteins and their aggregates. The first line of defence is the molecular chaperones which promote the correct folding of newly synthesised proteins and the refolding of incorrectly folded proteins (McClellan & Frydman 2001; Taylor et al. 2002; Gibbs & Braun 2008). In addition there are two main mechanisms; the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP), which remove misfolded proteins and aggregates through degradation (Rubinsztein 2006). However, in the case of neurodegenerative diseases these defence mechanisms may themselves be defective or are simply overwhelmed by the amount of misfolded protein and the progressive accumulation of aggregates and are unable to effectively prevent this harmful accumulation. This illustrates the difficulty in establishing cause and effect regarding the pathogenic mechanisms (Ciechanover & Brundin 2003) but either way the inefficiency of these defence mechanisms in preventing and removing misfolded proteins and aggregates could be argued to play a major role in the degeneration of neurons, although it remains a matter of debate. Developing therapeutics which target these defence mechanisms could provide effective strategies for treatment and prevention, i.e. by enhancing the defence mechanisms the misfolding of disease proteins and accumulation of aggregates could be significantly reduced.

It has been suggested that the accumulation of toxic protein aggregates can result from a single or a combination of the following pathological processes; "(1) abnormal synthesis and folding of disease proteins; (2) aberrant interactions of disease proteins with other proteins; (3) impaired degradation and turn over of disease proteins; (4) impaired intracellular transport of disease proteins, especially those targeted for axonal transport over long distances" (Roy et al. 2005).

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The impairment of axonal transport will be the focus of this review as axonal transport plays a vitally important role in the development and functioning of neurons (Chevalier-Larsen & Holzbaur 2006). Transport defects are therefore very likely to have significant adverse consequences on neuronal functioning and could thereby play a major part in neurodegenerative diseases. Much relatively recent broad-ranging research has shown that defects in axonal transport are a plausible mechanism of neurodegenerative diseases (reviewed by Roy et al. 2005; Chevalier-Larsen & Holzbaur 2006; El-Kadi et al. 2007). There are various cellular and molecular defects related to impaired axonal transport that not only represent an opportunity to gain a more complete understanding of these complex diseases, but also represent potential targets for the development of successful treatment and prevention strategies against these diseases.

It is still not clear whether protein aggregation and accumulation directly affects axonal transport through neurotoxic effects, or perhaps simply by creating a physical blockage, or if defects in axonal transport result in the accumulation of protein aggregates because it is difficult to establish cause and effect. Defects in axonal transport may enhance the accumulation of aggregates if they are not efficiently transported to the cell body for degradation (Chevalier-Larsen & Holzbaur 2006), which would further impair axonal transport, inevitably creating a viscous cycle leading to progressive degeneration of neurons. It therefore seems that regardless of it's context in terms of cause and effect of aggregate accumulation, axonal transport is likely to be a good target due to its great importance in the functioning of neurons.

Axonal transport

In humans, the vastly complex nervous system is divided into the central nervous system (CNS), consisting of the brain and the spinal cord which analyse, interpret and respond to incoming signals, essentially acting as a central control centre, and the peripheral nervous system (PNS). The PNS comprises the entire nervous system, excluding the brain and the spinal cord and allows communication between the CNS and the rest of the body. Neurons typically consist of a cell body, multiple dendrites and a single axon which branches into multiple nerve terminals (Alberts et al. 2004). In order to convey a signal from a sensory organ to the CNS and a subsequent response signal to activate for instance, a muscle in the foot, the neurons need to be of considerable length, with neurons reaching one metre or more in length (Gunawardena & Goldstein 2004).

The two major components which act as the 'rails' and the 'motors' of the axonal transport system (shown in Fig. 1) are the neuronal cytoskeleton and the molecular motors, respectively. The neuronal cytoskeleton provides structural support, whilst remaining dynamic to allow for required changes over time and it consists of three major components; microtubules, actin and intermediate filaments (Chevalier-Larsen & Holzbaur 2006). Long distance axonal transport primarily involves microtubules and transport also occurs along actin filaments. Microtubules are polarized structures with plus (at the synapse) and minus (at the cell body) ends and unidirectional molecular motors, kinesins and dyneins, are therefore associated with active transport in a specific direction; dyneins from plus to minus ends and kinesins from minus to plus ends (Gunawardena & Goldstein 2004).

Fig. 1  Microtubule-associated molecular motors transport a wide range of cargo along axons. Kinesins mediate anterograde transport and dyneins mediate retrograde transport (Figure obtained from Roy et al. 2005).

Efficiency of the axonal transport system is important as it transports many essential cargoes and thereby enables many vital functions to be fulfilled. However functioning at it's optimal level is of particular importance due to the extensive length of neurons.

Protein synthesis is mainly restricted to the neuronal cell body and axons are unable to synthesise the materials which they require for growth and repair. Therefore, once these essential axonal components have been synthesised in the cell body, they need to be actively transported to the location within the axon where required (Roy et al. 2005). The transport of molecules such as structural proteins and organelles away from the cell body to the synapse is known as anterograde transport, and associated with the previously mentioned kinesin molecular motors. Retrograde transport, is the complementary mechanism involved in axonal transport which transports cargo, such as neurotrophic factors in the opposite direction, i.e. away from the axon into the cell body, using dynein molecular motors (Charrin et al. 2005).

The cell body is also the primary site for degradation of misfolded and aggregated proteins (Chevalier-Larsen & Holzbaur 2006). Therefore another important function of axonal transport is to carry misfolded proteins to the cell body for degradation to avoid the accumulation of potentially toxic misfolded proteins and aggregates.

Early studies (Lasek 1968a; 1968b) using radiolabelled amino acids to investigate various aspects of axonal transport found that some proteins were transported rapidly, at rates of 100-400mm/day, whilst other proteins were transported at the significantly slower rate of approximately 0.2-5mm/day. It was not until relatively recently that the differences between these two components of axonal transport, termed fast and slow transport, were clarified. These more recent studies (Roy et al. 2000; Wang et al. 2000) using cultured neurons and labelled neurofilaments known to be moved by slow transport, showed slow axonal transport in real-time using epifluorescence microscopy. It was shown that the significantly decreased rate of transport observed in slow axonal transport is due to pauses in the movement of cargo rather than slower rates of transport and whilst the cargo is moving, it is moving at a 'fast' rate. It was estimated that the neurofilament cargo only spent 20% of the overall time moving and are therefore paused for the majority of the time (Roy et al. 2000). A unified perspective proposes that both fast and slow transport involve the same molecular motors, namely kinesins and dyneins, but the overall rate of transport differs due to the proportion of time spent moving (Brown 2003).

The cargo transported by the two components of axonal transport differ; fast axonal transport carries the "components of membranous organelles such as small vesicles, secretory granules, dense bodies, multivesicular bodies and mitochondria", whilst slow axonal transport carries "all the proteins of the cytoplasmic matrix including the cytoskeletal proteins and the soluble proteins of the axon" (Lasek et al. 1984) and there is currently not any evidence of membranous proteins being moved by slow axonal transport (Tytell et al. 1981).

The link between axonal transport and neurodegenerative diseases

The vital importance of axonal transport is emphasised as it is not only essential for transporting essential organelles such as mitochondria and transmitting the molecules involved in signalling throughout the body but also for the transport of the basic structural components of the nervous system, without which even the basic functioning of the complex nervous system would not be possible. This highlights the significance of the adverse effects which impairments in axonal transport could have on a range of functions throughout the body and it is therefore unsurprising that there is increasing evidence which clearly implicates defects in axonal transport in neurodegenerative diseases.

Roy et al. (2005) summarise the three recent developments which significantly implicate axonal transport in neurodegenerative diseases; "(1) the discovery of human motor protein mutations in neurodegenerative diseases, (2) axonal transport defects in animal and in vitro cellular models harboring human mutations, and (3) newly discovered roles for pathogenic proteins like amyloid precursor protein (APP), tau, presenilins and synucleins in the modulation and regulation of axonal transport" (Roy et al. 2005).

A variety of mutations affect various components, namely the 'rails' and 'motors' of the axonal transport system, which can impair and cause disruption of axonal transport. These defects are thought to lead to neurodegeneration through three main mechanisms; (1) the failure to supply newly synthesised axonal components needed for the growth and repair of the axon which would inevitably lead to axon degeneration, (2) the accumulation of toxic substances, such as aggregates of misfolded proteins in the axon, which could potentially cause a physical blockade in the axon, and (3) the inhibition of signalling pathways which would block essential communication throughout the body and could activate programmed cell death pathways (Chevalier-Larsen & Holzbaur 2006). Whether neurodegeneration is caused by a single or a combination of these mechanisms is still to be elucidated but there is a wide range of evidence of defects in various components of the axonal transport system which clearly implicate impaired axonal transport in the progressive degeneration of neurons in various degenerative diseases.

Mechanisms of disruption of axonal transport

The main mechanisms through which disruption of axonal transport arises, are caused by mutations in motor proteins and proteins which are involved in the neuronal cytoskeleton. Such mutations would inevitably have significant consequences on the overall functioning of axonal transport as they affect the two integral components, the 'rails' and the 'motors' of the axonal transport system. The complex interactions between the various components of this transport system mean that even a mutation in a single protein could have significant effects on the overall functioning of the system and therefore lead to neurodegeneration. It is therefore unsurprising that mutations in molecular motor proteins have been directly linked to various neurodegenerative diseases (reviewed by Roy et al. 2005), including various forms of motor neuron disease (Hafezparast et al. 2003; Puls et al. 2003; Puls et al. 2005), Charcot-Marie-Tooth disease Type 2A (Zhao et al. 2001; Tanaka & Hirokawa 2002) and Hereditary Spastic Paraplegia (Reid et al. 2002; Blair et al. 2006).

Molecular motors fulfil their function by moving a wide range and large volume of cargo along the microtubule 'rails' of the axonal transport system using energy generated by ATP hydrolysis and they are made up of two functional parts; a motor domain that converts this chemical energy into motion and interacts with specific elements of the neuronal cytoskeleton and a tail domain, which has evolved to allow motors to interact directly or indirectly via adaptor proteins with a wide range of cargo (Gunawardena & Goldstein 2004). There are two types of microtubule-dependent molecular motors, the kinesin superfamily proteins (KIFs) and the dynein superfamily proteins, whilst the myosin superfamily proteins are the actin-dependent molecular motors.

Molecular motors have many fundamental roles in a broad range of cellular processes, such as the important role that the microtubule-associated kinesins and dyneins play in neuronal development and neuronal regeneration (Hirokawa & Takemura 2004) and thus it is highly likely that mutations in genes encoding motor proteins will lead to progressive neurodegeneration.

The components of the axonal transport system are closely interlinked which includes coordination between the molecular motors. Kinesins are needed to transport both myosins (Huang et al. 1999) and dyneins (Ligon et al. 2004) to the synaptic end of the axon and without such transportation dyneins would not be able to fulfil their integral function as retrograde molecular motors (Hirokawa & Takemura 2004). This crucial coordination between the molecular motors illustrates how a mutation in a single molecular motor protein, in this case a kinesin, can have detrimental consequences on the entire axonal transport system.

Mutations in motor proteins

Kinesin mutations

The kinesins are the largest of the molecular motor superfamilies, comprising fourteen families (El-Kadi et al. 2007). They fulfil many important functions through mediating anterograde transport to supply the length of axon with essential components synthesised in the cell body and are vital in the coordination of the other molecular motors. As anterograde transport is responsible for delivering vital axonal components from the cell body to the entire length of an axon, it is likely that the longest axons will not only be the first but also the most severely affected by any defect in anterograde transport.

Kinesin-I was the first of these motors to be discovered (Vale et al. 1985) and consists of kinesin heavy chain (KHC) subunits, KIF5A, KIF5B and KIF5C (Xia et al. 1998), and kinesin light chain (KLC) subunits including KLC1, KLC2 and KLC3 (Rahman et al. 1998). Kinesin 'walks' along microtubules by a 'hand-over-hand' mechanism (Yildiz et al. 2004) and dynein has also been shown to use a similar, although a more irregular mechanism (Gennerich & Vale 2009). Many kinesins show cargo specificity and adaptor proteins are needed for the indirect attachment of many types of cargo to the molecular motor (Chevalier-Larsen & Holzbaur 2006) which again requires the coordination and full functioning of many different proteins to mediate efficient anterograde transport and emphasises the severe effect a single mutation in one part of this complex system could have on the overall functioning of axonal transport.

There is evidence which clearly implicates defects in axonal transport, caused by one of the anterograde microtubule-associated motors, in neurodegenerative diseases as it has been shown that Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by a mutation in KIF1Bβ, an isoform of the kinesin superfamily motor protein KIF1B, which transports synaptic vesicle precursors (Zhao et al. 2001). Charcot-Marie-Tooth disease (CMT) is a prevalent inherited early-onset neurological disease, with symptoms beginning in the first or second decade of life (Szigeti & Lupski 2009). The disease is characterised by peripheral neuropathy presenting clinical symptoms including the development of weakness and bony deformities in the feet, followed by weakness in the hands, loss of muscle stretch reflexes in the ankles, knees and upper limbs and mild sensory loss in the legs. In their study, Zhao et al. (2001) "generated kif1B heterozygous mice that mimic human CMT2A neuropathy" and found impaired axonal transport in these heterozygotes. As a result of the similarity between the disease in the mouse model and CMT2A in humans and the knowledge that the KIF1B locus has been mapped to the CMT2A disease interval (Gong et al. 1996), they analysed the KIF1B locus in human sufferers of CMT2A and found "a heterozygous A→T point mutation, which transformed Q98 to L" (Zhao et al. 2001). The Q98L mutation was shown to significantly affect the ATPase activity of the molecular motor, which would explain the impairment of axonal transport. Due to this evidence, clearly showing that the Q98L mutant KIF1Bβ protein subunit disrupted the function of the anterograde molecular motor and thereby significantly reduced the transport of cargo from the cell body to the peripheral axons which lack the ability to synthesise these crucial proteins, it is suggested "that a haploinsufficency of this motor protein is responsible for CMT2A neuropathy, both in this mouse model and in humans" (Zhao et al. 2001).

Further evidence has been provided to link defects in anterograde axonal transport to neurodegenerative diseases as it was shown that a missense mutation in the motor domain of the kinesin heavy chain (KIF5A) is present in a family with hereditary spastic paraplegia (HSP) (Reid et al. 2002). HSPs are a diverse heterogenous group of inherited genetic neurodegenerative disorders, "characterised clinically by progressive spasticity and weakness of the lower limbs, and pathologically by retrograde axonal degeneration" (Salinas et al. 2008). The KIF5A gene maps within the SPG10 interval originally identified as an autosomal dominant locus for pure HSP (Reid et al. 1999) and through the sequencing of 30 genes that mapped within this interval it was found that only the KIF5A gene exhibited a potential mutation. A subsequent study "identified an N256S missense mutation in the KIF5A gene in all affected family members in which the SPG10 locus was originally identified (Reid et al. 1999) but not in 220 normal control individuals" (Reid et al. 2002). "The N256S mutation occurs at an invariant asparagine residue...within the motor domain" (Reid et al. 2002) and such mutations have been shown to uncouple the binding of motors to microtubules, which prevents the activation of the motor ATPase by microtubules (Song & Endow 1998). This would have a significant negative effect on the functioning of the kinesin motor and thus impair the crucial anterograde transport of many vital cargoes from the cell body to destinations throughout the axon, which would inevitably lead to axon degeneration.

A missense mutation, also in the KIF5A gene associated with the SPG10 interval, has been found to cause adult-onset HSP with an average age of onset of 36 years, which is much later than previously mentioned cases of HSP (Blair et al. 2006). By sequencing the KIF5A gene they identified a missense mutation, Y276C, which causes a substitution of tyrosine for cysteine in exon 10 and was present in all the affected individuals in this study but not in any of the unaffected individuals or normal controls. Additional evidence from in silico analysis of the Y276C mutation indicating that the amino acid substitution has a damaging effect on the function of the protein supports the authors' conclusion that this mutation is the cause of this late-onset HSP.

Dynein and dynactin mutations

Through mediating retrograde transport, dynein fulfils many important functions including injury signalling, transporting misfolded proteins to the cell body to be degraded (Chevalier-Larsen & Holzbaur 2006) and this particular molecular motor has also been implicated in the autophagic clearance of misfolded protein (Ravikumar et al. 2005) which could all relate directly to the pathogenesis of neurodegenerative diseases. The majority of dynein functions require dynactin, a large multi-subunit complex which is proposed to provide the link between dynein and its cargo (Holleran et al. 1998). Two important subunits of dynactin are dynamitin (p50) and p150Glued.

Interesting findings have resulted from studying a family with an inherited slowly progressive form of motor neuron disease, distal spinal and bulbar muscular atrophy, which begins in the second or third decade of life and is characterised by early symptoms such as stridor (high-pitched whistling sound whilst breathing) resulting from vocal fold paralysis and later by weakness and atrophy in the face, distal legs and particularly hands (Puls et al. 2005). In an earlier study of this family, Puls et al. (2003) showed linkage of this disease to chromosome 2p13 and a single base-pair change at position 59 (G59S), which causes an amino-acid substitution of serine for glycine. This point mutation in the CAP-Gly motif of the p150Glued subunit of dynactin (DCTN1) causes the disease by impairing dynactin's microtubule-binding ability (Puls et al. 2003), thereby impairing dynein's ability to function as a retrograde motor which would have significant consequences on axonal transport. Analysis of the immunohistochemistry of this disease showed the accumulation of inclusions of dynein and dynactin in the hypoglossal motor neuron cell bodies and neurites (Puls et al. 2005). It is suggested by the authors that the degeneration of motor neurons could be due to either a shortage of neurotrophic factors transported by retrograde motors, which are essential for motor neuron survival, or to impairments in both retrograde and anterograde transport caused by the accumulation of cargo in the axon.

Dynamitin-overexpressing transgenic mice were used as a model in order to establish whether the targeted disruption of the retrograde molecular motor dynein is sufficient to cause the degeneration of motor neurons and associated symptoms of this group of neurodegenerative diseases (LaMonte et al. 2002). Overexpressing dynamitin is a particularly effective method to investigate the role of dynein in axonal transport and neurodegenerative diseases, as the overexpression of this subunit of dynactin has been shown to cause disruption of the dynein molecular motor complex and thereby disrupt axonal transport (Burkhardt et al. 1997). The mice in the study (LaMonte et al. 2002) demonstrated a loss of strength, reduction in nerve supply to the muscles and motor neuron degeneration, characterising a late-onset progressive motor neuron degenerative disease. The results obtained from this study clearly show that the overexpression of dynamitin results in the inhibition of retrograde transport which eventually results in the death of neurons, which confirms the critical role of impaired axonal transport in motor neuron degenerative diseases.

There is further evidence from a later study directly linking defects in axonal transport, caused by mutations in dynein, to motor neuron degenerative diseases (Hafezparast et al. 2003). This mouse model consisted of two mouse mutants Loa (Legs at odd angles) and Cra1 (Cramping 1), which are both "autosomal dominant traits that give rise to an age-related progressive loss of muscle tone and locomotor ability in heterozygous mice" (Hafezparast et al. 2003). Histopathological analysis of the disease in these mice showed that these mutations cause the progressive degeneration and a significant loss of α motor neurons in the spinal cord in both heterozygotes and homozygotes, and intracellular inclusions, similar to Lewy bodies, were found in homozygous mice. These mutations in the cytoplasmic dynein heavy chain 1 (Dnchc1) gene were shown to impair retrograde transport in homozygous Loa/Loa neurons as the frequency of high speed carriers appeared to be reduced and there was also an increase in pauses during transport.

A contrasting aspect of the role that dynein and retrograde transport play in the pathology of neurodegenerative diseases has also been the focus of research showing that the dynein/dynactin complex is required for the retrograde transport of misfolded protein in order for aggresome formation to occur (Johnston et al. 2002). This particular role of dynein is relevant to the pathology of a diverse range of neurodegenerative diseases, as protein aggresomes are a common characteristic of the majority of these diseases, specifically when considering aggresome formation as a neuroprotective mechanism. If aggresomes fulfil a protective role and mutations in dynein were to prevent the formation of such aggresomes, the symptoms and progression of such diseases may consequentially be even more severe. Another interesting aspect of this study is the finding that dynein is redistributed to aggresomes which could have significant implications in the impairment of axonal transport as there are likely to be adverse effects on retrograde transport of a variety of important cargo if the associated motors were not available.

Mutations affecting the neuronal cytoskeleton

As previously mentioned, the neuronal cytoskeleton forms the 'rails' of the axonal transport system which are essential for it's functioning and through this obligatory role the detrimental effect that defects in this central component of the system could have on the overall functioning and efficiency of axonal transport are highlighted.

Progressive motor neuropathy (pmn) mutant mice (Schmalbruch et al. 1991) which are frequently used as a model for human motor neuron disease, were used in a study to provide the first genetic evidence that defects in tubulin assembly can adversely affect microtubule assembly and thereby cause motor neuron degeneration (Bömmel et al. 2002). They "localised the genetic defect in pnm mice to a missense mutation in the tubulin-specific chaperone E (Tbce) gene affecting an evolutionary conserved amino acid residue" (Bömmel et al. 2002) which causes the substitution of tryptophan for glycine at position 524 of the important tubulin specific chaperone, CofE. This chaperone plays a vital role in the formation of the α- and β-tubulin heterodimeric complex, essential in the assembly of microtubules and this study provides evidence of the subsequent negative effect on axons as they are shorter and exhibit axonal swellings, which would undoubtedly have an adverse effect on axonal tranport. Another study using the same pmn mouse model provided further evidence that this missense mutation in the Tbce gene can cause motor neurodegenerative diseases (Martin et al. 2002).

A mutant small heat-shock protein, HSP27 was found to cause CMT and distal hereditary motor neuropathy (dHMN) and 5 distinct missense mutations in six unrelated families with either CMT2 or dHMN were identified (Evgrafov et al. 2004). Heat-shock protein chaperones, such as HSP27 fulfil important functions through their anti-apoptotic and cytoprotective roles and the discovery of this mutant in affected families is highly relevant in implicating defects in axonal transport in neurodegenerative diseases, as mutant HSP27 was shown to affect the assembly of neurofilaments, which would inevitably affect the structure of the neuronal cytoskeleton, which provides the 'rails' of the axonal transport system. Further support was provided by a later study (Ackerley et al. 2006), which found that mutations in HSP27 were responsible for a form of dHMN through the disruption of neurofilament assembly and axonal transport. This study showed that the mutant caused the formation of aggregates and the sequestration of essential components of the axonal transport system, such as the neurofilament middle chain subunit and the p150Glued subunit of dynactin, which would cause significant disruption to axonal transport.

Mutations in the gene encoding the neurofilament light chain (NEFL) were originally proposed to be the cause of CMT2E after this disease was studied in a single family (Mersiyanova et al. 2000) but this was not confirmed until further evidence was provided by the study of another family with this disease (De Jonghe et al. 2001), which identified a dominant double missense mutation in the NEFL gene, which causes an amino acid substitution of proline with arginine and is thereby thought to destabilise the NEFL, which is highly likely to affect the microtubule 'rails' of the axonal transport system.


Mutations in either of the microtubule-associated molecular motors and in components of the neuronal cytoskeleton are clearly sufficient to cause a range of neurodegenerative diseases. Discussed studies clearly illustrate that subtle mutations, causing for example a single amino acid substitution in a single gene can have severely detrimental effects on the entire axonal transport system and therefore cause widespread neurodegeneration throughout the body, which is to be expected as the mutant genes encode integral components of the 'rails' and the 'motors' in a transport system in which all the components are closely interlinked.

In many of the neurodegenerative diseases discussed, the neurons in the distal extremities of the body are often the first and most severely affected, with these parts of the body showing the first clinical symptoms of the disease. It is likely that these distal neurons are most at risk due to their extensive length, as even a slight impairment in axonal transport would severely affect these extremities as newly synthesised components and other essential cargo such as mitochondria would not reach these parts of the axon. In addition signalling pathways would be impaired, which would inevitably lead to the degeneration of these axons and therefore cause extensive neurodegeneration.

These findings highlight the high potential of defects in axonal transport as key targets for therapeutics and a rapidly increasing understanding, not only of the causes but also the complex interactions between the components and interrelated mechanisms of these diseases. They provide an optimistic outlook regarding progress in terms of developing successful approaches for the effective treatment and prevention of neurodegenerative diseases.

As the development and progression of neurodegenerative diseases often appear to be caused by multiple pathogenic mechanisms, perhaps an effective way to progress would be to develop treatment strategies which target a combination of these mechanisms, i.e. target the mutant genes which cause axonal transport defects whilst also enhancing chaperone activity to prevent protein misfolding, and removal mechanisms to prevent the accumulation of aggregates, as this would prevent, or at least limit the viscous cycle which is likely to cause the full onset of neurodegenerative diseases.