Chapter Four - Calpain in the cleavage of alpha-synuclein and the pathogenesis of Parkinson's disease
Introduction
Parkinson's disease (PD) is a complex, chronic progressive neurodegenerative disorder for which there is no cure.1 Impacting approximately 6.3 million people in the world, this devastating disease is considered one of the most prevalent neurodegenerative motor disorders. During disease progression, neurons in the spinal cord (SC) and substantia nigra (SN) are damaged, leading to impaired motor function in the patient.2, 3 As a result, patients may manifest devastating symptoms including postural instability, bradykinesia, and resting tremors, which drastically lower the quality of life for the patient.1, 4 l-DOPA therapy is widely used but it shows temporary relief and can worsen the patients' symptoms if it is used for an extended period of time. Thus investigating the mechanisms involved in disease progression may help develop therapeutic strategies for the improvement of quality of life in PD.
Identified during disease prognosis are pathological features of PD, many of which are shared similarities among other neurodegenerative diseases, including rapid loss of dopaminergic neurons in the SN and, interestingly, accumulations of Lewy-bodies in the brain.5 Lewy-bodies are toxic filaments and are composed of a small, lipid-binding protein called synuclein.4 Alpha-synuclein (α-synuclein), a 140 amino acid-long protein, is abundant in the nervous system and is predominately expressed in neurons, even in healthy individuals, comprising 10% of cystolic protein.6 When mutated, the α-synuclein protein may become truncated. Previous studies have correlated this truncated, mutated form of α-synuclein with the harmful Lewy-body aggregation and consequential cellular toxicity. A study using transgenic mice implicates α-synuclein C-terminus mutations with reduced dopamine in the striatum. These data, in turn, suggest toxicity of the Lewy-bodies in disease pathology.7 It is not well known how the α-synuclein accumulations occur,8, 9 and the mechanisms promoting disease progression are also not well characterized.
α-Synuclein may promote a disruption of calcium homeostasis. Calcium is a key and versatile agent in many vital biological systems, and this powerful ion acts as a valuable physiological messenger for transport across the plasma membrane and activation of essential enzymes, among other functions. Ionic transport using Na+ and K+ utilizes an approximate 10- to 30-fold gradient across the cellular membrane. However, Ca2 +-powered transport is driven by a steep 20,000-fold concentration difference between the extracellular and intracellular space, affirming calcium's role as a valuable signal for responding to rapid-changing extracellular and intracellular conditions. By mediating calcium transport, cells can activate or inhibit Ca2 +-dependent signaling pathways, and neurons are especially sensitive to the effects provided by calcium for initiating response. Therefore, normal calcium homeostasis is necessary for improved biological function.4
Perturbation of Ca2 + homeostasis may activate calpain, a neutral protease, setting off a cascade of defense responses, as suggested by previous research.4 Due to this dysregulation of Ca2 + homeostasis and calpain activation, the immune system, in turn, responds with the activation and migration of inflammatory T cells (CD4 + and CD8 +), microglial activation, and astrocyte immobilization, features particularly studied in PD and other neurodegenerative diseases.4, 10, 11
Microglia activation is particularly interesting. Microglia are endogenous neural cells known as brain macrophages and are responsible for innate immunity in the brain. Classified under a family of immune cells, called the Major Histocompatibility Complex Class 2 (MHC-II), they can initiate responses leading to inflammation and neuron death.12 Cells also relevant to inducing inflammation are chemokines and cytokines. Their primary function involves recruiting leukocytes to sites of inflammation. Their respective receptors are upregulated by calpain activation, therefore upregulating chemokine and cytokine molecules. The dysregulation of these molecules is often associated with PD.12
Recently, in addition to dopaminergic degeneration in the striatum as observed in PD,1, 5 damage to non-dopaminergic sites, namely, the spinal cord, has been demonstrated. The mechanisms describing how degeneration occurs both in the brain and in the spinal cord remain elusive. It has been suggested, however, that inflammation may be a key factor to this nervous system degeneration, and interestingly, neuro-inflammation has been implicated to promote the brain's susceptibility to neurodegeneration.2, 13
The induced cell death following calpain activation is of particular interest to our laboratory. We suggest several hypotheses for the mechanisms following calpain activation and subsequent initiation of neuron death. The first hypothesis is that calpain will directly activate microglia. Microglial will secrete factors that will activate T cells. We also propose that calpain-activated T cells will produce neuroinflammatory factors, which may further activate microglia to produce factors killing neurons. Activated microglia may process α-synuclein generating peptides for T cell activation. Processing of α-synuclein by activated microglia may also produce toxic synuclein peptides causing neuronal cell death. Further, the damaged neurons by above processes may release detrimental factors that will activate and/or kill naïve immune cells and neurons, and thus perpetuating this vicious cycle for continued progression of the disease (Fig. 1).
Section snippets
Calpain
Calpain is an intracellular Ca2 +-dependent cysteine protease that is widely distributed and show regulated protease activity at neutral pH.14, 15, 16 Calpain is comprised of two subunits: an 80 kDa subunit called the “μ-calpain large” (μCl) subunit and a regulatory 30 kDa subunit.16, 17 The large subunit contains four domains. The functions of the first and third domains are not yet well characterized. It is known, however, that the fourth domain is a Ca2 +-binding domain. Moreover, as the main
α-Synuclein
α-Synuclein, a 140 amino acid-long protein, is translated from the SNCA gene.43 The α-synuclein protein belongs to a family of proteins found in vertebrates called the synucleins, including also β-synuclein and γ-synuclein, classified for their small size, solubility, and having a highly-conserved α-helix lipid-binding motif.44
α-Synuclein protein contains three distinct regions. At residues 1–60, there is a six-repeat polyprotein lipid-binding motif at the amino terminus. At residues 61–95,
Microglial activation, neuroinflammation, and neurodegeneration in PD
Microglia are neural immune cells, and they account for approximately 12% of all the cells in the brain.54, 55, 56 They are structurally interesting. In healthy neural tissue, they have a small, circular cell body with branching projections, and this is considered to be its ramified state. Microglia in ramified morphology plays several crucial roles in the nervous system, including synaptic plasticity, axon growth, determination for neuron fate, and migration.56, 57, 58 They are additionally
T cell activation, neuroinflammation, and neurodegeneration in PD
A vital agent of the acquired immune system is the T cell. The CD4 + T cell is a variety of T cells heavily implicated with PD and the associated neurodegeneration. Once activated by antigen-presenting cells (APCs), the T cells will undergo phenotypic changes dependent on nearby mediating factors. For example, T cells may differentiate into a T-helper 1 phenotype when in the presence of IL-12. The diverse phenotypes that the T cells can possess suggest a specialization for target pathogens. Of
Calpain cleavage of α-synuclein and presentation by microglia and professional APCs
Both calpain and endogenous calpain inhibitor calpastatin are localized in the cytosol, and take part in selective protein cleavage in response to calcium signaling.75 Alterations in calcium homeostasis may lead to calpain activation and contribute to CNS disorders. Under abnormal calcium signaling, calpastatin can be degraded by calpain, allowing calpain to escape from inhibition by calpastatin. This could be problematic because of insufficient regulation of calpain-mediated proteolysis in
Cross presentation of synuclein peptides to T cells
The brain's blood brain barrier is a border protecting the brain from invasion of foreign proteins. For this reason, it was previously understood that the blood brain barrier plays as a separation between the brain and the rest of the body. However, it has been suggested that the brain directly communicates with the immune and endocrine systems.79 Therefore, when these systems elicit systemic inflammatory reactions and responses, the brain is influenced and can change brain function. Similarly,
Conclusions
Alterations in calcium homeostasis may lead to calpain activation. We believe differential conditions of calpain activation can contribute to our knowledge of PD disease progression. Particularly interesting is calpin's role in microglial activation. The duality of microglia activation, promoting protection with short term activation and degradation with long term activation, gives hints for understanding the disease's complexity. As one of its damaging roles, microglia cells maintain
Acknowledgments
This study was made possible by grant from the South Carolina Spinal Cord Injury Research Funds (SCIRF#2016 I-03 and SCIRF #2018 I-01) to A.H. Contents do not necessarily represent the policy of the SCIRF and do not imply endorsement by the funding agency. This work was also supported by grants from the Ralph H. Johnson Veterans Administration Medical Center, Charleston (BX004269, 1I01BX002349-01) to N.L.B.
Conflicts of interest
The authors have no financial conflicts of interest.
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