We would like to comment on the interesting results of the recent study by Morfini et al. (1). Kinesin-1, a major microtubule motor that transports cargo in the plus-end direction of microtubules, is a heterotetramer consisting of two microtubule-binding, motor polypeptides (the heavy chains; KHCs) and two cargo-binding polypeptides (the light chains; KLCs). Being a soluble, cytoplasmic protein, kinesin-1 needs to bind the cargo in order to transport it. Therefore, recruitment of kinesin-1 to the cargo vesicle, and its release from it, are important regulatory steps of axonal transport. About 10 years ago, Scott Brady’s laboratory identified the first mechanism leading to the release of kinesin-1 from vesicles. According to this model, kinesin-1 is released through the action of the chaperone HSC70, and is nucleotide-dependent and NEM-sensitive (2). One year later, work from Larry Goldstein’s laboratory suggested that the premature release of kinesin-1 from cargo vesicles in neurons could impair fast axonal transport and lead to neuronal pathology and disease (3). Although the...  Read more

We would like to comment on the interesting results of the recent study by Morfini et al. (1). Kinesin-1, a major microtubule motor that transports cargo in the plus-end direction of microtubules, is a heterotetramer consisting of two microtubule-binding, motor polypeptides (the heavy chains; KHCs) and two cargo-binding polypeptides (the light chains; KLCs). Being a soluble, cytoplasmic protein, kinesin-1 needs to bind the cargo in order to transport it. Therefore, recruitment of kinesin-1 to the cargo vesicle, and its release from it, are important regulatory steps of axonal transport. About 10 years ago, Scott Brady’s laboratory identified the first mechanism leading to the release of kinesin-1 from vesicles. According to this model, kinesin-1 is released through the action of the chaperone HSC70, and is nucleotide-dependent and NEM-sensitive (2). One year later, work from Larry Goldstein’s laboratory suggested that the premature release of kinesin-1 from cargo vesicles in neurons could impair fast axonal transport and lead to neuronal pathology and disease (3). Although the mechanisms for the release of kinesin-1 from its vesicular cargos were incompletely understood at that time, the general idea that a premature release of the motor from its cargo could be at the core of the pathology in neurodegenerative diseases turned out to be correct, and generated an increased interest for research in this direction. Thus, work from the Brady and Busciglio laboratories identified at least two pathways for release of kinesin-1 from vesicles and halt of transport, which are likely to be factors leading to the axonal pathology and synaptic failure in Alzheimer’s disease (AD) (4-6).

Both pathways lead to phosphorylation of the KLCs, followed by detachment of kinesin-1 from the cargo, and impairment of vesicle transport. They are initiated by the addition of soluble Aβ oligomers, or expression of FAD-linked presenilin 1 variants, which trigger aberrant activation of casein kinase 2 or glycogen synthase 3β, which phosphorylate the KLCs. Why the phosphorylated kinesin-1 is released from vesicles is still not fully understood.

Along with AD, kinesin-1 is a target for abnormal phosphorylation in other neurodegenerative diseases, such as spinal and bulbar muscular atrophy (SBMA) and Huntington’s disease, as revealed by the studies from the Brady laboratory, including the work featured here (1, 7). However, in this case, the phosphorylation targets the KHCs, and the activated kinase that performs the phosphorylation is the cJun-N-terminal kinease (JNK). The phosphorylation of the KHCs leads to inhibition of binding of kinesin-1 to microtubules. As a result, the kinesin-1-cargo complex is released from the microtubules, and the transport is halted. These studies showed that the abnormal activation of JNK is triggered by the pathogenic, polyglutaminated, mutant proteins characteristic for polyglutamine (polyQ) expansion diseases: polyQ-androgen receptor in SBMA) (7), and polyQ-huntingtin in Huntingon’s disease(1). As the study by Morfini et al. (1) showed, polyQ-huntingtin activates JNK3, a neuron-specific JNK, that in turn phosphorylates KHC at a serine residue critical for the microtubule-binding function of kinesin-1. While in this case JNK3 is aberrantly activated by a disease factor, it is likely that, under normal conditions, the JNK-3 pathway contributes to the regulation of axonal transport.

Interestingly, in the squid axon system used in these studies, polyQ-huntingtin inhibits, not only the anterograde (kinesin-driven), but also the retrograde (cytoplasmic dynein-driven) fast axonal transport (1). It is not clear whether this inhibition of transport in both directions is due to the fact that kinesin-1 and cytoplasmic dynein interact and coordinate each other’s function (8), or is caused by a direct effect on the dynein machinery. Other studies showed that huntingtin regulates dynein-mediated vesicle transport, and can interact with both dynein and its accessory complex, dynactin (9, 10); however, the assays used by Morfini et al. (1) did not detect an interaction of huntingtin with dynein.

Certainly, other mechanisms, besides the release of the kinesin motor from the cargo or the microtubules, could contribute to the pathogenic processes in these neurodegenerative diseases. Other potentially damaging pathways that target the intracellular transport by affecting the cytoskeleton or the supply of ATP (by disrupting mitochondrial function) have been described (reviewed in (11)). Also, the activation of the kinases is likely to lead to the abnormal phosphorylation of other protein targets as well, with detrimental consequences for the function of neurons via mechanisms that may not involve abnormal axonal transport. For now, a picture emerges where the release of kinesin-1 from either cargo or microtubules, followed by impairment of axonal transport, becomes an important component of the pathogenic process in many neurodegenerative diseases. Therefore, it is the time to think of possibilities to correct the deficiencies, or to find means to enhance the disease-inflicted axonal transport.

References:
1. Morfini GA, You YM, Pollema SL, Kaminska A, Liu K, Yoshioka K, Björkblom B, Coffey ET, Bagnato C, Han D, Huang CF, Banker G, Pigino G, Brady ST. Pathogenic huntingtin inhibits fast axonal transport by activating JNK3 and phosphorylating kinesin. Nat Neurosci. 2009 Jul;12(7):864-71. Abstract

2. Tsai MY, Morfini G, Szebenyi G, Brady ST. Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport. Mol Biol Cell. 2000 Jun;11(6):2161-73. Abstract

3. Kamal A, Almenar-Queralt A, LeBlanc JF, Roberts EA, Goldstein LS. Kinesin-mediated axonal transport of a membrane compartment containing beta-secretase and presenilin-1 requires APP. Nature. 2001 Dec 6;414(6864):643-8. Abstract

4. Morfini G, Szebenyi G, Elluru R, Ratner N, Brady ST. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility. EMBO J. 2002 Feb 1;21(3):281-93. Abstract

5. Pigino G, Morfini G, Atagi Y, Deshpande A, Yu C, Jungbauer L, Ladu M, Busciglio J, Brady S. Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta. Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5907-12. Abstract

6. Pigino G, Morfini G, Pelsman A, Mattson MP, Brady ST, Busciglio J. Alzheimer's presenilin 1 mutations impair kinesin-based axonal transport. J Neurosci. 2003 Jun 1;23(11):4499-508. Abstract

7. Morfini G, Pigino G, Szebenyi G, You Y, Pollema S, Brady ST. JNK mediates pathogenic effects of polyglutamine-expanded androgen receptor on fast axonal transport. Nat Neurosci. 2006 Jul;9(7):907-16. Abstract

8. Ligon LA, Tokito M, Finklestein JM, Grossman FE, Holzbaur EL. A direct interaction between cytoplasmic dynein and kinesin I may coordinate motor activity. J Biol Chem. 2004 Apr 30;279(18):19201-8. Abstract

9. Caviston JP, Ross JL, Antony SM, Tokito M, Holzbaur EL. Huntingtin facilitates dynein/dynactin-mediated vesicle transport. Proc Natl Acad Sci U S A. 2007 Jun 12;104(24):10045-50. Abstract

10. Zala D, Colin E, Rangone H, Liot G, Humbert S, Saudou F. Phosphorylation of mutant huntingtin at S421 restores anterograde and retrograde transport in neurons. Hum Mol Genet. 2008 Dec 15;17(24):3837-46. Abstract

11. De Vos KJ, Grierson AJ, Ackerley S, Miller CC. Role of axonal transport in neurodegenerative diseases. Annu Rev Neurosci. 2008;31:151-73. Abstract

View all comments by Zoia Muresan
View all comments by Virgil Muresan

Huntington disease (HD) is an ultimately fatal genetic neurodegenerative disease with a triad of cognitive, neuropsychiatric, and motor symptoms caused by the polyglutamine expansion in the coding region of the Huntingtin gene. Despite intense research in the field, there are currently no disease-modifying treatments for HD and treatment remains limited to management of symptoms. Since the discovery of the gene, the search for anatomical or molecular explanations for the preferential loss of striatal medium spiny neurons in HD has be an active area of research. Mutant huntingtin is expressed throughout the body, so the reason that it should preferentially lead to loss of a small subset of neurons is not obvious.

Several hypotheses regarding the preferential involvement of the striatum in the course of HD pathogenesis have emerged over the past decade. In 2001, Zuccato et al. suggested that a lack of neurotrophic support from BDNF, normally made in the cortex and transported to the striatum, is at least in part responsible for this preferential susceptibility. In 2002, Zeron...  Read more

Huntington disease (HD) is an ultimately fatal genetic neurodegenerative disease with a triad of cognitive, neuropsychiatric, and motor symptoms caused by the polyglutamine expansion in the coding region of the Huntingtin gene. Despite intense research in the field, there are currently no disease-modifying treatments for HD and treatment remains limited to management of symptoms. Since the discovery of the gene, the search for anatomical or molecular explanations for the preferential loss of striatal medium spiny neurons in HD has be an active area of research. Mutant huntingtin is expressed throughout the body, so the reason that it should preferentially lead to loss of a small subset of neurons is not obvious.

Several hypotheses regarding the preferential involvement of the striatum in the course of HD pathogenesis have emerged over the past decade. In 2001, Zuccato et al. suggested that a lack of neurotrophic support from BDNF, normally made in the cortex and transported to the striatum, is at least in part responsible for this preferential susceptibility. In 2002, Zeron et al. provided evidence for a role for NR2B-subtype NMDAR activation as a trigger for selective neuronal degeneration in HD. More recent studies (Benchoua, 2008; Chavrin, 2005) have suggested that the high expression of dopamine exacerbates mutant huntingtin toxicity in the striatum.

In this study, Subramaniam and colleagues describe a series of elegant biochemical experiments showing that a recently identified, striatally-enriched small G protein, Rhes (ras homolog enriched in the striatum), directly interacts with Huntingtin, induces SUMOylation of the mutant protein, and thereby confers mutant Huntingtin toxicity to cells expressing it. Previous studies have implicated Rhes in dopamine signaling and striatal function, with conflicting reports on the effect of Rhes knockout on locomotion in different genetic backgrounds (Errico, 2008; Spano, 2004). Their findings also suggest that Rhes might cause toxicity, partly, by preventing neurons from forming the intracellular deposits of mutant Htt called inclusion bodies. These results are consistent with previous findings indicating that inclusion body formation may serve as a protective response by neurons against more soluble and toxic forms of the protein (Arrasate et al., 2004).

The current study provides an interesting insight into the selectivity of cell death in Huntington disease. Preventing farnesylation of Rhes may be a therapeutic target to investigate, especially since farnesyl transferase inhibitors have already been developed for the clinic. The absence of drastic phenotypes in the knockouts is encouraging, if Rhes is to be pursued as a therapeutic target.

However, further studies are needed to truly establish Rhes as a striatal-specific mediator of mutant Huntington toxicity and as a therapeutic target for HD therapy. First and foremost, it will be essential to verify that Rhes mediates toxicity of mutant Huntingtin in primary neurons, in vivo, as well as in the cell line models used in this study. Will the HD phenotype be significantly reduced in a Rhes knockout background? Will ectopic expression of Rhes in primary cortical and hippocampal neurons confer the same degree of toxicity of mutant Huntington as in primary striatal neurons? Second, while Rhes is highly enriched in the striatum, its mRNA is also expressed in various other regions, notably the CA1/CA3 layers of the hippocampus, the granular layer of the cerebellum, and anterior thalamus (Vargiu 2004, Harrison 2008). Whether the Rhes protein is also expressed in these other neuronal population needs further exploration to determine whether Rhes is a truly striatal-specific factor. Third, both Rhes and dopamine are suggested to contribute to the preferential loss of striatal neurons, and Rhes is suggested to have roles in dopamine signaling in the striatum. Finally, it will be interesting to see whether Rhes can also explain the differential susceptibilities of medium spiny neurons and interneurons within the striatum, as interneurons are largely spared in HD.

As with other aspects of biology, a complex image is developing regarding the factors that regulate the preferential loss of striatal neurons in HD. Whether these anatomical and molecular regulators of toxicity, described here or yet to be discovered, act in a concerted fashion or independent of each other remains to be explored. Rhes is already suggested to have roles in dopamine signaling, and both Rhes and dopamine are suggested to contribute to the selective vulnerability of striatal neurons. How these signaling pathways are regulated and fine-tuned within a complex neural network will give us a better understanding of the pathological basis in Huntington disease.

References:
Arrasate M. et al., 2004. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431:805–810. Abstract

Benchoua A. et. al., 2008. Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II. Hum Mol Genet 17:1446–1456. Abstract

Charvin D., et. al., 2005. Unraveling a role for dopamine in Huntington's disease: the dual role of reactive oxygen species and D2 receptor stimulation. PNAS. 102:12218–12223. Abstract

Errico F. et. al., 2008. The GTP-binding Protein Rhes Mdulates Dopamine Signaling in Striatal Medium Spiny Neurons. Mol Cell Neurosci. 37: 335-345. Abstract

Falk J. D. et. al., 1999. Rhes: A Striatal-Specific Ras Homolog Related to Dexras1. J. Neurosci Res. 57: 782-788. Abstract

Harrison L. M. et. al., 2008. Ontogeny and Dopaminergic Regulation in Brain of Ras Homolog Enriched in Striatum (Rhes).Brain Res. 1245: 16-25. Abstract

Spano D. et. al., 2004. Rhes Is Involved in Striatal Function. Mol and Cell Bio, 24(13): 5788-5796. Abstract

Sharp A. H. et. al., 1995. Widespread Expression of Huntington's Disease Gene (IT15) Protein Product. Neuron, 14: 1065-1074. Abstract

Vargiu P. et. al., 2004. The Smalle GTP-binding Protein, Rhes, Regulates Signal Transduction from G protein-Coupled Receptors.

Zeron M. M. et. al., 2002. Increased sensitivity to N-methyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of Huntington's disease. Neuron, 33: 849-860. Abstract

Zuccato C. et. al., 2001. Loss of huntingtin-mediated BDNF gene transcription in Huntington's disease. Science,293: 493-498. Abstract

View all comments by Steven Finkbeiner
View all comments by Hengameh Zahed

These two reports from Scott Brady’s and Larry Goldstein’s laboratories are highly significant because they extend the concept that neurodegenerative disease is caused by impaired axonal transport, beyond more common disorders like Alzheimer's, to also include triplet-repeat diseases. The implication is that multiple neurodegenerative diseases may share a similar mechanism. This notion was proposed nearly 20 years ago by Carlton Gajdusek, but many years went by before sufficient technical advances occurred in AD research to provide circumstantial and experimental data supporting this view. Traction in this area began with the demonstration that tau (a microtubule binding protein) was the building block of AD neurofibrillary tangles (NFTs). Also helpful was the resolution of the controversy over the role of NFT formation in AD in 1991 by studies showing that abnormally phosphorylated CNS tau proteins (PHFtau) form the paired helical filaments in AD NFTs, and that excessive phosphorylation of PHFtau reduced its...  Read more

These two reports from Scott Brady’s and Larry Goldstein’s laboratories are highly significant because they extend the concept that neurodegenerative disease is caused by impaired axonal transport, beyond more common disorders like Alzheimer's, to also include triplet-repeat diseases. The implication is that multiple neurodegenerative diseases may share a similar mechanism. This notion was proposed nearly 20 years ago by Carlton Gajdusek, but many years went by before sufficient technical advances occurred in AD research to provide circumstantial and experimental data supporting this view. Traction in this area began with the demonstration that tau (a microtubule binding protein) was the building block of AD neurofibrillary tangles (NFTs). Also helpful was the resolution of the controversy over the role of NFT formation in AD in 1991 by studies showing that abnormally phosphorylated CNS tau proteins (PHFtau) form the paired helical filaments in AD NFTs, and that excessive phosphorylation of PHFtau reduced its ability to bind microtubules (MTs) and stabilize them in order to support axonal transport. For a detailed review of this research area, see Lee et al., 2001.

Thus, years before it was discovered that loss of tau function was the consequence of tau gene mutations in hereditary tauopathies, such as frontotemporal dementia with parkinsonism linked to chromosome 17 or FTDP-17, it was already appreciated that wild-type tau, when altered by hyperphosphorylaton in AD, sustained a loss of function that might impair axonal transport and so lead to a neurodegenerative disease. This prompted the hypothesis that the generation of PHFtau depletes neurons of tau able to bind microtubules, thereby leading to brain degeneration in AD. This model predicted that: 1) the conversion of tau into PHFtau disrupts MT-based transport as well as perhaps physically “blocking” transport due to accumulations of PHFs in neurons and their processes, and 2) the failure of neurons to export proteins from the cell body to distal processes and to retrieve substances (e.g., trophic factors) internalized at axon terminals compromises neuronal viability. It was proposed that these events would culminate in neuronal dysfunction and degeneration leading to the onset/progression of AD. Remarkably, nearly all of the predictions of this disease model of tau pathology in AD and related tauopathies were validated in the last four years through studies of tau-transgenic mice. Some of these provided experimental proof that neurodegeneration caused by tau aggregation was linked to axonal transport failure (Isihara et al., 1999). Indeed, a consensus in favor of this notion appears to be building and a whole issue of (Neuromolecular Medicine) was dedicated to this topic last year.

It will be important to confirm and extend the findings described in these two studies, which differ in some details. Both papers conclude that impaired axonal transport plays a significant role in mechanisms underlying neurodegeneration. Significantly, the views proposed in these papers complement and extend the earlier concept of a loss of function and impairment of axonal transport when tau is altered in AD, FTDP-17, and other tauopathies. Specifically, the authors of both of these Neuron papers propose that polyQ species acquire a toxic gain of function that disrupts axonal transport. By adding a toxic gain of function in disease proteins to the more well-documented loss of normal function (as in hyperphosphorylated tau), and linking these abnormalities to impaired axonal transport, these two studies open up bold new avenues for advancing insights into mechanisms of neurodegenerative disease. All of this could have substantial implications for the discovery of new and better therapies for AD and other less common neurodegenerative diseases such as Huntington’s, FTDP-17, other tauopathies, and related disorders.

View all comments by John Trojanowski

I find this research compelling. I have always maintained that basic cell/molecular/genetic biology would lead the way to the aberrant processes in signal transduction. The interface of biochemistry will, of course, better describe and predict the appropriate chemical environmental milieu, which accompanies such complex cell dynamics; however, being able to observe maladapted (stressed, aged, epigenetically modified form-function) but otherwise normally present cell proteins, signalers, and molecular switches will be of great aide to correcting problems that have negative/positive feedback loops provided by nature.

It is through knowing the proper roles, points of transport/processing and what goes wrong in the system that we may devise appropriate therapeutic tools and targets.

References:
Genes 7. Harrisons Principles of Internal Medicine.

View all comments by Jacob Mack

This article reiterates the critical importance of studying AD in the context of the neuron/brain as a whole, and also underlines the fact that the neuron is different from every other cell in our body.

View all comments by Subhojit Roy

The study by Pigino et al. study elegantly highlights a possible mechanism by which Aβ oligomers can influence axonal transport. Though the validity of intracellular Aβ is debatable in the context of human AD pathology, Pigino et al. convincingly show that in a simple model-system of axonal transport, nanomolar levels of Aβ can influence transport; they also provide convincing evidence for the involvement of a specific signaling cascade in this process. The paper is a must-read!

View all comments by Subhojit Roy

This paper by Subramaniam and colleagues presents some intriguing findings. For one thing, they identify the small G protein, Rhes, as defining a potential new class of non-traditional SUMO E3 ligases, thus opening a potential new window on the SUMOylation machinery. In addition, their study raises the possibility that Rhes activity may exacerbate the pathology of mutant Htt by preferentially causing its SUMOylation with consequences similar to those observed in Drosophila and cells. It will be interesting to see whether Rhes knockout mutations will suppress pathogenesis in mouse models of HD.

View all comments by J. Lawrence Marsh