Binding of neuronal α-synuclein to β-III tubulin and accumulation in a model of multiple system atrophy

doi:10.1016/j.bbrc.2011.12.092

Biochemical and Biophysical Research Communications

Volume 417, Issue 4, 27 January 2012, Pages 1170-1175

https://doi.org/10.1016/j.bbrc.2011.12.092 Get rights and content

Abstract

Multiple system atrophy (MSA) is a neurodegenerative disease caused by α-synuclein (α-syn) accumulation in oligodendrocytes and neurons. We generated a transgenic (Tg) mouse model in which human α-syn was overexpressed in oligodendrocytes. Our previous studies have revealed that oligodendrocytic α-syn inclusions induced neuronal α-syn accumulation, thereby resulting in progressive neuronal degeneration in mice. We also demonstrated that an insoluble complex of α-syn and β-III tubulin in microtubules progressively accumulated in neurons, thereby leading to neuronal degeneration. In the present study, we demonstrated that neuronal accumulation of the insoluble complex was derived from binding of α-syn to β-III tubulin and not from α-syn self-aggregation. Thus, interaction between α-syn and β-III tubulin plays an important role in neuronal α-syn accumulation in an MSA mouse model.

Highlights

► Binding of α-synuclein to β-III tubulin causes neuronal degeneration in MSA model. ► α-Synuclein interacted with β-III tubulin and formed an insoluble protein complex. ► Rifampicin reduces α-synuclein self-aggregation in GCI formation. ► Suppression of microtubule polymerization prevents neuronal α-synuclein accumulation. ► Microtubule depolymerization repressed the accumulation by binding to β-III tubulin.

Introduction

Multiple system atrophy (MSA) is a non-hereditary neurodegenerative disease clinically characterized by various combinations of parkinsonism, cerebellar dysfunction, and autonomic nervous system failure [1], [2]. Two significant pathological features that characterize MSA are glial cytoplasmic inclusions (GCIs) and neuronal inclusions, both of which are composed of α-synuclein (α-syn). GCIs, the first neuropathological manifestation of MSA to be described, are oligodendrocytic inclusions [3], [4], [5], [6]. α-Syn accumulation forms a major component of MSA inclusions [7], [8] and may be the primary lesion that eventually compromises nerve cell function and viability [9]. However, cellular mechanisms underlying neurodegeneration are not completely understood, and unfortunately, no approach has had an immediate therapeutic impact on the treatment of MSA neurodegeneration. We previously generated a transgenic (Tg) mouse model in which human wild-type α-syn was overexpressed under the control of the 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) promoter in oligodendrocytes in the mouse central nervous system (CNS) [10]. Our study of this CNP-α-syn Tg mouse model demonstrated that formation of oligodendrocytic α-syn inclusions resulted in neuronal degeneration characterized by a phenotype of motor impairment, macroscopically apparent brain atrophy, and histologically decreased neuronal number with gliosis [10]. α-Syn accumulation in oligodendrocytes induced its neuronal accumulation and caused progressive neuronal degeneration in Tg mice. We hypothesized that a similar disease process underlies MSA. Accordingly, we studied novel mechanisms of neuronal α-syn accumulation in the MSA mouse model and identified the protein microtubule β-III tubulin, which interacts with α-syn to form an insoluble protein complex that progressively accumulates in neurons, thereby leading to neuronal dysfunction [11]. In addition, we demonstrated that accumulation of the insoluble α-syn complex was suppressed by treatment with nocodazole, a microtubule-depolymerizing agent. α-Syn, which predominantly exists at presynaptic terminals in CNS, has a tendency to aggregate in amyloid fibrils in vitro [12], [13]. α-Syn self-aggregation is crucial for the pathogenesis of neurodegeneration in synucleinopathy. Rifampicin, an antibiotic, has been reported to inhibit α-syn self-aggregation in vitro and cause disaggregation of fibrils in a concentration-dependent manner [14]. To investigate direct interaction of α-syn contributing to neuronal accumulation of insoluble α-syn in the MSA mouse model, we examined the effect of rifampicin on α-syn accumulation in primary cultured cells derived from mice. We compared the effect of rifampicin with that of nocodazole, a microtubule-depolymerizing agent that suppresses insoluble α-syn accumulation. Simple inhibition of α-syn self-aggregation by rifampicin did not decrease neuronal α-syn accumulation. We hope to clarify the essential role of binding of α-syn to β-III tubulin in neuronal α-syn accumulation and thereby provide evidence for potential therapeutic targets in the treatment of MSA.

Section snippets

Expression of α-syn and β-III tubulin

Complete cDNAs of mouse α-syn and mouse β-III tubulin genes (Scna and Tubb3, respectively) were cloned into pET30a(+) (Novagen, CA, USA), which carries a His-tag in the N-terminal sequence. Scna and Tubb3 were expressed in Escherichia coli BL21(DE3)pLysS cells (Novagen). The cells were suspended in 20 mM Tris–HCl and 0.5% Triton X-100 at pH 7.4 and disrupted by ultrasonication. The cell lysate was centrifuged at 12,000 × g for 30 min at 4 °C. For Tubb3 purification, the pellet was resuspended in 20

Role of β-III tubulin in α-syn aggregation in vitro

To understand the role of β-III tubulin in insoluble α-syn accumulation, we inhibited binding of α-syn to β-III tubulin and assessed α-syn accumulation in vitro. Cloned cDNAs of Snca and Tubb3 were expressed in E. coli, and recombinant proteins were purified. α-Syn aggregation was produced by recombinant α-syn alone or by recombinant α-syn in the presence of β-III tubulin and monitored by ThT, a fluorescent dye that interacts with amyloid fibrils, during incubation of the two recombinant

Discussion

In the MSA mouse model, oligodendrocytic accumulation of α-syn induced secondary neuronal accumulation of insoluble α-syn, thereby leading to neurodegeneration [10]. Using primary cultured cells derived from the mouse brain, we demonstrated that α-syn interacted with β-III tubulin and formed an insoluble protein complex [11]. The insoluble complex of α-syn and β-III tubulin in microtubules progressively accumulated in neurons. In the present study, although rifampicin was found to decrease

Acknowledgments

We thank V. Lee and J. Trojanowski for providing MSA Tg mice. This work was supported by the Research Funding for Longevity Sciences (23–11) from the National Center for Geriatrics and Gerontology and Grants-in-Aids for Scientific Research (22500326 and 22700386) from the Japan Society for the Promotion of Science.

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Thus, the strategy of preventing their formation or sequestering these species has the potential to be the correct approach. Interestingly, among the players that could influence the aggregation processes there is the microtubule system (Alim et al., 2002; Esteves, 2010; Kim et al., 2008; Nakayama et al., 2012; Outeiro et al., 2007). This might suggest that targeting the interplay between α-synuclein and microtubules could prevent α-synuclein aggregation.
Looking at the puzzle that depicts the molecular determinants in neurodegeneration, many pieces are lacking and multiple interconnections among key proteins and intracellular pathways still remain unclear. Here we focus on the concerted action of α-synuclein and the microtubule cytoskeleton, whose interplay, indeed, is emerging but remains largely unexplored in both its physiology and pathology. α-Synuclein is a key protein involved in neurodegeneration, underlying those diseases termed synucleinopathies. Its propensity to interact with other proteins and structures renders the identification of neuronal death trigger extremely difficult. Conversely, the unbalance of microtubule cytoskeleton in terms of structure, dynamics and function is emerging as a point of convergence in neurodegeneration. Interestingly, α-synuclein and microtubules have been shown to interact and mediate cross-talks with other intracellular structures. This is supported by an increasing amount of evidence ranging from their direct interaction to the engagement of in-common partners and culminating with their respective impact on microtubule-dependent neuronal functions. Last, but not least, it is becoming even more clear that α-synuclein and tubulin work synergically towards pathological aggregation, ultimately resulting in neurodegeneration. In this respect, we supply a novel perspective towards the understanding of α-synuclein biology and, most importantly, of the link between α-synuclein with microtubule cytoskeleton and its impact for neurodegeneration and future development of novel therapeutic strategies.
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Oligodendrocytes are in contact with neurons, wrap axons with a myelin sheath that protects their structural integrity, and facilitate nerve conduction. Oligodendrocytes also form a syncytium with astrocytes which interacts with neurons, promoting reciprocal survival mediated by activity and by molecules involved in energy metabolism and trophism. Therefore, oligodendrocytes are key elements in the normal functioning of the central nervous system. Oligodendrocytes are affected following different insults to the central nervous system including ischemia, traumatism, and inflammation. The term oligodendrogliopathy highlights the prominent role of altered oligodendrocytes in the pathogenesis of certain neurological diseases, not only in demyelinating diseases and most leukodystrophies, but also in aging and age-related neurodegenerative diseases with abnormal protein aggregates. Most of these diseases are characterized by the presence of abnormal protein deposits, forming characteristic and specific inclusions in neurons and astrocytes but also in oligodendrocytes, thus signaling their involvement in the disease. Emerging evidence suggests that such deposits in oligodendrocytes are not mere bystanders but rather are associated with functional alterations. Moreover, operative modifications in oligodendrocytes are also detected in the absence of oligodendroglial inclusions in certain diseases. The present review focuses first on general aspects of oligodendrocytes and precursors, and their development and functions, and then introduces and updates alterations and dysfunction of oligodendrocytes in selected neurodegenerative diseases with abnormal protein aggregates such as multiple system atrophy, Lewy body diseases, tauopathies, Alzheimer’s disease, amyotrophic lateral sclerosis, frontotemporal lobar degeneration with TDP-43 inclusions (TDP-43 proteinopathies), and Creutzfeldt-Jakob´s disease as a prototypical human prionopathy.
The role of transcriptional control in multiple system atrophy
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For instance, the presence of Al3+ and Cu2+ may induce structural perturbation (Paik et al., 1997, 1999), and point mutations at A30P, E49K, and A53T were shown to reduce protein hydrophobicity (Li et al., 2001), thus leading to the formation of nonfunctional and deadly aggregates (Uversky, 2007). In addition, the interaction between α-synuclein and β-III tubulin in the transgenic mouse model and the nitration of α-synuclein tyrosine residues are critical for the formation of insoluble protein complexes that progressively accumulate in neurons (Ischiropoulos and Beckman, 2003; Nakayama et al., 2012; Souza et al., 2000). These accumulated α-synuclein complexes lead to neuronal dysfunction (Nakayama et al., 2009).
Multiple system atrophy (MSA) is an α-synucleinopathy that is clinically characterized by varying degrees of parkinsonian, autonomic, and cerebellar features. Unlike other α-synucleinopathies such as Parkinson's disease, MSA is unique in that the principal α-synuclein lesions, called glial cytoplasmic inclusions, occur in oligodendroglia rather than neurons, with significantly more α-synuclein accumulating in MSA brain compared with Parkinson's disease. Although well defined clinically, the molecular pathophysiology of MSA has barely been investigated. In particular, there have been no systematic studies of the perturbation of the brain transcriptome during the onset and progression of this disease. Interestingly, measurements of α-synuclein gene (SNCA) expression in MSA brain tissue have not revealed overexpression of this gene in oligodendroglia or neurons. It has therefore become clear that other genes and gene networks, both directly as noncoding RNAs or through protein products, contribute to the accumulation of the α-synuclein protein in the brain. This review provides a summary of current developments in the investigation of the transcriptional causes of MSA and outlines perspectives for future research toward the elucidation of the molecular pathology of MSA-specific neurodegeneration.
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Multiple system atrophy (MSA) is a fatal adult-onset neurodegenerative disorder of uncertain etiopathogenesis manifesting with autonomic failure, parkinsonism, and ataxia in any combination. The underlying neuropathology affects central autonomic, striatonigral and olivopontocerebellar pathways and it is associated with distinctive glial cytoplasmic inclusions (GCIs, Papp-Lantos bodies) that contain aggregates of α-synuclein. Current treatment options are very limited and mainly focused on symptomatic relief, whereas disease modifying options are lacking. Despite extensive testing, no neuroprotective drug treatment has been identified up to now; however, a neurorestorative approach utilizing autologous mesenchymal stem cells has shown remarkable beneficial effects in the cerebellar variant of MSA. Here, we review the progress made over the last decade in defining pathogenic targets in MSA and summarize insights gained from candidate disease-modifying interventions that have utilized a variety of well-established preclinical MSA models. We also discuss the current limitations that our field faces and suggest solutions for possible approaches in cause-directed therapies of MSA.
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Multiple system atrophy is an intractable neurodegenerative disease caused by α-synuclein (α-syn) accumulation in oligodendrocytes and neurons. With the use of a transgenic mouse model overexpressing human α-syn in oligodendrocytes, we demonstrated that oligodendrocytic α-syn inclusions induce neuronal α-syn accumulation, resulting in progressive neuronal degeneration. The mechanism through which oligodendrocytic α-syn inclusions trigger neuronal α-syn accumulation leading to multiple system atrophy is unknown. In this study, we identified cystatin C, an oligodendrocyte-derived secretory protein that triggers α-syn up-regulation and insoluble α-syn accumulation, in neurons of the mouse central nervous system. Cystatin C was released by mouse oligodendrocytes overexpressing human α-syn, and extracellular cystatin C increased the expression of the endogenous α-syn gene in wild-type mouse neurons. These neurons then accumulate insoluble α-syn and may undergo apoptosis. Cystatin C is a potential pathogenic signal triggering neurodegeneration in multiple system atrophy.
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It was also reported that α-syn binds to monomeric tubulin and stimulates microtubule polymerization (24). Indeed, blocking tubulin polymerization using nocodazole resulted in reduced α-syn accumulation (12, 16). Furthermore, ingestion of nocodazole attenuated impaired synaptic function in MSA mice (13).
Multiple system atrophy (MSA) is a neurodegenerative disease caused by α-synuclein aggregation in oligodendrocytes and neurons. Using a transgenic mouse model overexpressing human α-synuclein in oligodendrocytes, we previously demonstrated that oligodendrocytic α-synuclein inclusions induce neuronal α-synuclein accumulation and progressive neuronal degeneration. α-Synuclein binds to β-III tubulin, leading to the neuronal accumulation of insoluble α-synuclein in an MSA mouse model. The present study demonstrates that α-synuclein co-localizes with β-III tubulin in the brain tissue from patients with MSA and MSA model transgenic mice as well as neurons cultured from these mice. Accumulation of insoluble α-synuclein in MSA mouse neurons was blocked by the peptide fragment β-III tubulin (residues 235–282). We have determined the α-synuclein-binding domain of β-III tubulin and demonstrated that a short fragment containing this domain can suppress α-synuclein accumulation in the primary cultured cells. Administration of a short α-synuclein-binding fragment of β-III tubulin may be a novel therapeutic strategy for MSA.

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Binding of neuronal α-synuclein to β-III tubulin and accumulation in a model of multiple system atrophy

Abstract

Highlights

Introduction

Section snippets

Expression of α-syn and β-III tubulin

Role of β-III tubulin in α-syn aggregation in vitro

Discussion

Acknowledgments

Orthostatic hypotension and nicotine sensitivity in a case of multiple system atrophy

J. Neurol. Neurosurg. Psychiat.

Second consensus statement on the diagnosis of multiple system atrophy

Neurology

Glial cytoplasmic inclusions in the CNS of patients with multiple system atrophy (striatonigral degeneration, olivopontocerebellar atrophy and Shy-Drager syndrome)

J. Neurol. Sci.

NACP/α-synuclein immunoreactivity in fibrillary components of neuronal and oligodendroglial cytoplasmic inclusions in the pontine nuclei in multiple system atrophy

Acta Neuropathol. (Berl)

Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple system atrophy brains contain insoluble α-synuclein

Ann. Neurol.

Filamentous α-synuclein inclusions link multiple system atrophy with Parkinson’s disease and dementia with Lewy bodies

Neurosci. Lett.

The α-synucleinopathies: Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy

Ann. NY Acad. Sci.

Neuropathology of synuclein aggregates

J. Neurosci. Res.

Alpha-synuclein and neurodegenerative diseases

Nat. Rev. Neurosci.

Mouse model of multiple system atrophy: α-synuclein expression in oligodendrocytes causes glial and neuronal degeneration

Neuron