Particle radiation-induced dysregulation of protein homeostasis in primary human and mouse neuronal cells

Abstract

Space particle radiations may cause significant damage to proteins and oxidative stress in the cells within the central nervous system and pose a potential health hazard to humans in long-term manned space explorations. Dysregulation of the ubiquitin-proteasome system as evidenced by abnormal accumulation of polyubiquitin (pUb) chain linkages has been implicated in several age-related neurodegenerative disorders by mechanisms that may involve the inter-neuronal spread of toxic misfolded proteins, the induction of chronic neuroinflammation, or the inappropriate inhibition or activation of key enzymes, which could lead to dysfunction in, for example, proteolysis, or the accumulation of post-translationally-modified substrates.In this study, we employed a quantitative proteomics method to evaluate the impact of particle-radiation induced alterations in three major pUb-linked chains at lysine residues Lys-48 (K-48), Lys-63 (K-63), and Lys-11 (K-11), and probed for global proteomic changes in mouse and human neural cells that were irradiated with low doses of 250 MeV proton, 260 MeV/u silicon or 1 GeV/u iron ions. We found significant accumulation in K-48 linkage after 1 Gy protons and K-63 linkage after 0.5 Gy iron ions in human neural cells. Cells derived from different regions of the mouse brain (cortex, striatum and mesencephalon) showed differential sensitivity to particle radiation exposure. Although none of the linkages were altered after proton exposure, both K-48 and K-63 linkages in mouse striatal neuronal cells were elevated after 0.5 Gy of silicon or iron ions. Changes were also seen in proteins commonly used as markers of neural progenitor and stem cells, in DNA binding/damage repair and cellular redox pathways. In contrast, no significant changes were observed at the same time point after proton irradiation. These results suggest that the quality of the particle radiation plays a key role in the level, linkage and cell type specificity of protein homeostasis in key populations of neuronal cells.

Introduction

The brain contains many different functional regions with widely varying sensitivity to radiation. Among the more sensitive regions are those that are rich in progenitor cell types. A well-studied example is the dentate gyrus region of the hippocampus, where even relatively low-dose irradiation causes a significant but transient increase in the number of apoptotic progenitor cells (Yang et al., 2010). Consequences of radiation exposures in human radiotherapy patients have shown that, while the acute effects of brain irradiation such as somnolence, headache and memory defects are usually temporary, late-onset effects can be severe and irreversible. For example, survivors of childhood leukemia who had undergone prophylactic whole-brain irradiation were found to have a >6-fold increased risk of stroke (Bowers et al., 2006). The delayed complication of radiation-induced brain necrosis, which is related to damage to the vascular epithelium, is another serious effect that can occur >1 year after radiotherapy for brain tumor. A number of compelling studies over the years have provided us with a great deal of knowledge about the central nervous system (CNS) effects of particle radiation and has been carefully reviewed in the recent NCRP Report No 183 (NCRP, 2019). In particular, effects on rodent behavior (Denisova et al., 2002; Rabin et al., 1998, 2012, 2007) and cognitive abilities (Britten et al., 2012; Joseph et al., 1992; Lonart et al., 2012; Raber et al., 2004, 2011; Shukitt-Hale et al., 2003, 2004) have been well documented through the years. The results of heavy ion particle irradiation on cognitive impairment and increased Aβ plaque pathology have been reported in a double transgenic mouse model of AD (Cherry et al., 2012). Among the molecular hallmarks of several neurodegenerative diseases, those manifesting from dysfunction of protein homeostasis in neurons resulting in the accumulation of ubiquitin-positive protein aggregates, are particularly prominent. Studies of the detailed molecular mechanisms of radiation-induced CNS damage have linked the oxidative damage produced by radiation exposure to pathways affected in long-term, slowly progressing neurodegenerative diseases. For example, sustained CNS inhibition of cellular autophagy, a process important for neuronal protein homeostasis, has been shown to occur after HZE irradiation (Poulose et al., 2011). The goals of our studies are to further investigate radiation-quality dependent signaling mechanisms associated with protein homeostasis in brain cells.

Ubiquitin is a small (76 amino acids) protein that is known to mediate several important cellular functions through a well-controlled and tightly orchestrated ubiquitin proteasome system (UPS). In its canonical role, ubiquitin is covalently attached as a polyubiquitin chain to proteins, targeting them for proteolytic degradation by the 26S proteasome complex in an ATP-dependent process (Hershko, 1997). Protein modification by ubiquitin is a highly regulated process catalyzed by a series of three enzyme families known as E1, E2, and E3, which are capable of modifying the correct protein in the correct cellular location at the correct time in order to control important processes such as cell cycle progression, removal of damaged or mis-folded proteins, and gene transcription. It is now known that ubiquitin has other roles in addition to tagging proteins for proteasome degradation. The cellular pools of ubiquitin consist of (1) polyubiquitin chains attached to proteins, (2) monoubiquitin adducts on proteins, (3) linear precursor protein or (4) those that are attached to activating transferase and ligase enzymes. There is a great deal of complexity in the ubiquitin-mediated signaling mechanism (Ravid and Hochstrasser, 2008). For example, while polyubiquitin chains are linked to a protein through the Lys-48 (K-48), the presence of ubiquitin is the classical signal for proteasome degradation of the target protein. Modification of proteins with polyubiquitin chains linked through ubiquitin Lys-63 (K-63) is involved in receptor endocytosis and intracellular trafficking, and mono-ubiquitylation of histones is involved in regulating the structure of chromatin. Lys-11 (K-11) regulates DNA damage-induced transcription silencing in an ATM-dependent manner, is responsible for the assembly of K-11 linkage conjugates on damaged chromatin and is distinctly different from the K-63 linkages (Paul and Wang, 2017). Other reports also suggest possible biological roles for extracellular ubiquitin, including signaling through chemokine receptors on leukocytes and variety of cell types (Saini et al., 2010a, 2010b).

There are multiple factors that control ubiquitylation of specific proteins in the cell. Among these are the N-terminus amino acids, which have been shown to influence the intracellular stability of proteins, where various amounts of enzymatic and non-enzymatic processing can convert stable proteins into unstable ones by triggering their rapid ubiquitylation and degradation. N-terminal amino acids that confer instability include the basic residues arginine, lysine, and histidine, as well as the large hydrophobic amino acids leucine, isoleucine, phenylalanine, tryptophan, and tyrosine. N-terminal acidic amino acids aspartic acid, glutamic acid and cysteic acid (from oxidation of a cysteine residue) can be enzymatically modified by the ligation of an arginine residue onto the N-terminus in a process that is catalyzed by the enzyme arginyl-transferase, thereby rendering the protein unstable. The importance of this pathway in disease development has been well established.

The critical role of the ubiquitin proteome-system in memory is well recognized in the neuroscience literature (Jarome and Devulapalli 2018; Lip et al., 2017; Orsi et al., 2019). Recent quantitative analysis of brain cells and tissues from Alzheimer's disease (AD) patients has shown significant increases in pUb linkages as well as in the number of the ubiquitylation sites compared to those from age-matched negative controls (Abreha et al., 2018). Characterized ubiquitin-positive pathological protein aggregates in samples from 5 AD patients were compared to those obtained from 5 age-matched controls. Differential enrichment analysis showed that >800 ubiquitylation sites were significantly altered (∼80% increased) in AD samples, including 7 pUb linkages. In the microtubule-associated protein Tau, a core component of neurofibrillary tangles, the number of pUb sites increased most strongly in AD samples. Results suggest cross-talk between phosphorylation and ubiquitylation occurs on Tau in AD. Justified by this emerging role of ubiquitylation in aging related CNS diseases, we predicted that quantitative changes in cellular ubiquitin pools occur when neuronal cells are exposed to particle-radiation.

In this paper, we report our first quantitative assessment of particle-radiation induced alteration in cultured primary human and mouse neuronal stem cells. We quantified levels of three major pUb linkages, namely K11, K48, and K63 as well as global protein expressions using high-resolution mass-spectrometry based methods. Our results revealed particle beam fluencies, radiation dose, and cell-type dependent changes in the levels of intracellular ubiquitin and subsequent downstream effects on regulation of stress-associated signaling pathways. For NASA, one of the important concerns with long-duration missions outside the Earth's magnetic fields is the potential for charged particle exposure to cause short or long-term CNS dysfunction in astronauts (NCRP, 2019). The results of our findings on the mechanisms of protein processing in brain cells after particle radiation and linking these to neurodegenerative diseases will be useful to NASA to design of appropriate countermeasures to ameliorate any potential negative effects in space flights.