Elsevier

Neuroscience Letters

Volume 704, 21 June 2019, Pages 89-94
Neuroscience Letters

Research article
The metastability of the proteome of spinal motor neurons underlies their selective vulnerability in ALS

https://doi.org/10.1016/j.neulet.2019.04.001Get rights and content

Highlights

  • Spinal motor neuron proteome is supersaturated relative to oculomotor neurons.

  • Proteins corresponding to genes downregulated in ALS are more supersaturated.

  • Proteins encoded by genes upregulated in ALS are less supersaturated.

  • Ion channels and transporters are supersaturated and downregulated.

Abstract

Amyotrophic lateral sclerosis (ALS) is a heterogeneous motor neuron disease with familial forms linked to numerous mutations in a range of genes. The resulting variant proteins, including SOD1, TDP-43, and FUS, disturb protein homeostasis in a variety of ways and lead to the formation of intracellular inclusion bodies that are characteristic of different neuropathological subtypes of the disease. These inclusions are made up of scores of proteins that do not appear at first to share obvious characteristics other than coaggregation. Recent evidence, however, suggests that these aggregating proteins can be characterized as being supersaturated in spinal motor neurons, as they exhibit cellular concentrations exceeding their solubilities. Here, we show that the average supersaturation of the entire spinal motor neuron proteome is greater than that of the ALS-resistant oculomotor neurons, suggesting that the vulnerability of spinal motor neurons is linked to the overall metastability of their proteome against aggregation. Consistently, ALS expression data suggest that affected neurons respond to pathology by transcriptional downregulation of supersaturated proteins, including specifically ion channels. These results identify a mechanism by which protein homeostasis imbalance leads to inclusion body formation in ALS, and to a disruption of other processes dependent on proteins that are supersaturated, thereby resulting in the dysfunctional excitability alterations observed in vivo.

Introduction

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder in which the selective loss of upper and lower motor neurons in the motor cortex and spinal cord leads to impairment of muscle control, paralysis and eventually death. Protein aggregation and inclusion formation is associated with all forms of ALS, suggesting that protein misfolding is a common feature of the various forms of ALS [[1], [2], [3]]. In this respect, ALS is similar to other neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and Huntington’s diseases [[4], [5], [6]], which are also characterised by the formation of aberrant protein deposits.

While ˜90-95% of ALS cases are sporadic (sALS) and of unclear cause, the remainder of cases are inherited (familial ALS, or fALS) and can be linked to specific genetic mutations. Mutations in one or more of at least a dozen genes give rise to fALS, with most resulting in the aggregation of TDP-43, while in forms where TDP-43 pathology is absent FUS or SOD1 aggregates are present. In the context of ALS, the protein aggregate load correlates with areas of neuronal loss in the spinal cord [2,[7], [8], [9], [10]], and with cell death in culture [11], consistent with the idea that protein aggregates are intimately linked with motor neuron cell death. Recent work also suggests that disease progression may be a result of a prion-like propagation of protein misfolding and aggregation throughout the nervous system [[12], [13], [14]]. While the precise reason for inclusion formation to be associated with most ALS cases is unclear, it is apparent that protein homeostasis is perturbed [15].

Protein aggregates consisting of a wide range of proteins are increasingly recognized as being common to a range of neurodegenerative diseases, an observation attributable to the fact that even in their native states many proteins can be unstable towards aggregation [5,[16], [17], [18]]. To understand why some proteins aggregate in disease states whereas others remain soluble, we recently observed that many proteins in the proteome exceed their solubilities at the level at which they are expressed [19], that is, they are supersaturated [20,21]. In the specific context of ALS, we found that the combination of a spinal motor neuron expression profile and a high supersaturation score can explain many key features of the disease-specific protein inclusion fingerprint [19]. In addition, we recently showed that the mutant SOD1 induced alterations in ubiquitin homeostasis were partly explained by an increase in ubiquitylation of supersaturated proteins [22]. Previous work has found that across neurodegenerative disorders more generally, including Alzheimer’s and Parkinson’s diseases, proteins in major disease-associated pathways, as well as those that coaggregate within inclusion bodies, tend to be supersaturated [21]. It has also been recently shown, in the case of Alzheimer’s disease, that the characteristic progression of pathology across brain tissues is recapitulated by a protein expression signature in healthy brains of aggregation-prone proteins [23], which is also responsible for the selective vulnerability of specific neuron types [24]. Collectively these data are consistent with the notion that protein homeostasis breakdown and inability of the cell to deal with supersaturated proteins is associated with neurological disorders [5,20,21,23,24].

Recently, it has been proposed that the downregulation of supersaturated proteins in Alzheimer’s disease may limit their aggregation in response to compromised protein homeostasis [25]. In the present study, we examined experimental information acquired from expression analysis of vulnerable motor neurons in healthy and diseased tissue [[26], [27], [28]]. We aimed specifically to determine the relationship between protein supersaturation, cell-specific vulnerability and the transcriptional changes that occur during ALS. We found distinct differences in supersaturation between resistant oculomotor neurons and vulnerable spinal motor neurons. Moreover, genes downregulated in ALS generally correspond to metastable proteins at risk of aggregation, as they are supersaturated, while those that are upregulated correspond to proteins that are within their solubility limits. In the long term, however, while the downregulation of supersaturated proteins may represent a mechanism to limit aggregation, the chronic decrease of vital proteins such as ion channels may in turn lead to neuronal dysfunction and ultimately death.

Section snippets

Identification of co-aggregating proteins

Co-aggregating proteins in ALS were identified as in [19]. Proteins were only included if published data clearly showed co-localisation in human post-mortem tissue.

Identification of axonal channels and transporters

Previous work using proteomics identified axonal proteins from rat neuronal primary cultures [29]. A list of all axonal channels, pumps and transporters was generated from that of all identified axonal proteins.

Calculation of Zyggregator scores

Zyggregator scores were calculated as described in [21].

Calculation of supersaturation scores

Motor neuron specific supersaturation scores were calculated for

Vulnerable spinal motor neurons have a metastable proteome

We have shown previously that ALS inclusions are formed by proteins that tend to be supersaturated under physiological conditions [19]. These particular proteins were found to be distinguished from the proteins that form the functional network of normal interaction partners of the ALS-associated proteins SOD1, TDP-43 and FUS by their supersaturation levels when calculated using expression values in motor neurons, but not when averaged over several tissues.

Here we investigated whether the

Discussion

It has been recently reported that the proteins associated with ALS inclusions tend to be metastable to aggregation because they are supersaturated, specifically in motor neurons [19]. Here we have found that the supersaturation level of the entire proteome differentiates vulnerable from resistant motor neurons, and observed that a cellular response to the intrinsic metastability of the proteome is the transcriptional downregulation of supersaturated genes. In support of these conclusions, we

Acknowledgements

L.O. is supported by a National Health and Medical Research Council (NHMRC) of Australia Boosting Dementia Research Leadership Fellowship (APP1135720). C.M.D. and M.V. are members of the Cambridge Centre for Misfolding Diseases and were supported by the Wellcome Trust. J.J.Y. was supported by grants from the NHMRC (Grants 1095215 and 1084144), and Motor Neuron Disease Research Institute of Australia (Betty Laidlaw Prize).

References (38)

  • F. Chiti et al.

    Protein misfolding, functional amyloid, and human disease

    Annu. Rev. Biochem.

    (2006)
  • T.P. Knowles et al.

    The amyloid state and its association with protein misfolding diseases

    Nat. Rev. Mol. Cell Biol.

    (2014)
  • B. Bolognesi et al.

    ANS binding reveals common features of cytotoxic amyloid species

    ACS Chem. Biol.

    (2010)
  • M.T. Giordana et al.

    TDP-43 redistribution is an early event in sporadic amyotrophic lateral sclerosis

    Brain Pathol.

    (2010)
  • J. Brettschneider et al.

    TDP-43 pathology and neuronal loss in amyotrophic lateral sclerosis spinal cord

    Acta Neuropathol.

    (2014)
  • N. Ticozzi et al.

    Protein aggregation and defective RNA metabolism as mechanisms for motor neuron damage

    Cns Neurol. Disord. - Drug Targets

    (2010)
  • P.N. Leigh et al.

    Ubiquitin-immunoreactive intraneuronal inclusions in amyotrophic lateral sclerosis. Morphology, distribution, and specificity

    Brain

    (1991)
  • L. McAlary et al.

    Susceptibility of mutant SOD1 to form a destabilized monomer predicts cellular aggregation and toxicity but not in vitro aggregation propensity

    Front. Neurosci.

    (2016)
  • J.I. Ayers et al.

    Experimental transmissibility of mutant SOD1 motor neuron disease

    Acta Neuropathol.

    (2014)
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