Increased ISGylation in Cases of TBI-Exposed ALS Veterans

Abstract

Veterans who have served in the military are at a nearly 60% greater risk of being diagnosed with amyotrophic lateral sclerosis (ALS). Literature reports suggest that a history of traumatic brain injury (TBI) may be a risk factor for ALS in veterans. However, no diagnostic biomarkers are available for identifying ALS risk/development in TBI-exposed veterans. Here, using a Wes assay, we show that ISGylation, a conjugated form of interferon-stimulated gene 15 protein, is significantly elevated in the lumbar spinal cords (SC-Ls) of TBI-ALS compared with ALS veterans without a previous history of TBI (nonTBI-ALS). Although not as striking as in TBI-ALS veterans, ISGylation is also increased in nonTBI-ALS compared with normal veterans. Notably, no changes in ISGylation were seen in occipital lobe samples obtained from the same patients, suggesting that elevated ISGylation is distinct to ALS disease-specific SC-Ls. Moreover, we detected increased ISGylation in cerebral spinal fluid samples of TBI-ALS veterans. Other results using cultured lymphocyte cell lines show a similar trend of increased ISGylation in ALS patients from the general population. Together, these data suggest that ISGylation could serve as a diagnostic biomarker for TBI-ALS veterans, nonTBI-ALS veterans, and nonveterans affected by ALS.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a rare, incurable, and fatal neurodegenerative disease that affects upper motor neurons in the brain and lower motor neurons in the brainstem and spinal cord (1). Collectively, both upper and lower motor neurons control voluntary muscle movements of the body such as chewing, walking, breathing, and talking (1). Atrophy of these motor neurons, therefore, causes the muscles under their control to weaken, leading to the loss of all voluntary body functions, ultimately resulting in paralysis. ALS can affect individuals of any age, but it mostly affects late-/middle-aged individuals (45–55 years) and can be familial (hereditary) or sporadic (spontaneous due to environmental conditions). Approximately 5%–10% of ALS cases are familial, and 90% are sporadic, suggesting the contribution of environmental conditions as a predominant cause of ALS. Remarkably, research supported by the Department of Veterans Affairs and the Department of Defense has revealed that veterans who have served in the military are at a nearly 60% greater risk of being diagnosed with ALS than those with no history of military service (2). An independent study conducted by investigators at the Harvard University and Institute of Medicine supports these conclusions. However, what causes the disease in normal individuals and why the incidence of ALS is higher among veterans is not known. Literature reports suggest that a history of traumatic brain injury (TBI) may be a risk factor for ALS in veterans (3–5). However, a biochemical link between TBI and ALS is not known. Other reports demonstrating that interferon-stimulated gene 15 (ISG15), a protein of interest in our lab, is elevated in ALS patients (6) and in response to TBI in mice (7) suggests a plausible link between ISG15-TBI-ALS and led us to investigate ISGylation in TBI-exposed ALS veterans.

ISG15 is a ubiquitin-like protein that is minimally expressed in human normal cells and tissues. However, its gene and protein expressions are highly elevated in response to type I interferons (IFNs) in all cell lineages (8). ISG15 protein is synthesized from the ISG15 gene, and either remains in an intracellular free form, appended to proteins in cells (conjugated form), or secreted from cells (extracellular form) by an unknown mechanism (9). ISG15-specific enzymes E1 (UbE1L), E2 (UbcH8), and E3 (HERC5, EFP, and several others) are also IFN-stimulated proteins that conjugate intracellular free ISG15 to cellular proteins, a mechanism referred to as ISGylation (9). Empirical evidence from our lab has revealed that ISGylation predominantly antagonizes the canonical ubiquitin pathway in cancer (10) and ataxia telangiectasia (A-T) (11), a rare neurodegenerative disease. Since polyubiquitylation of cellular proteins is a prerequisite for protein turnover via the 26S proteasome, and ubiquitin-mediated protein turnover is crucial in maintaining cellular homeostasis, ISG15 proteinopathy (ISG15-mediated defective protein turnover) is proposed to be an underlying cause of malignancy (10, 12) and A-T neurodegeneration (11, 13) in human and mouse experimental disease models.

Like A-T, the IFN pathway is also aberrantly expressed in the spinal cords of affected mice in an ALS murine model (6). Free ISG15 is also elevated in the spinal cords of human ALS patients (6). However, whether ISGylation is elevated and induces proteinopathy in human ALS patients has not been investigated. Notably, ISG15 levels are increased in the brains of mice subjected to TBI (7). TBI due to blast explosions, motor vehicle accidents, and gunshot wounds during war is commonly seen in veterans. TBI damages neurons and ISG15 has been identified as a biomarker for neuronal injury (14). However, whether ISG15 and ISGylation are induced in TBI-exposed veterans diagnosed with ALS is not known, a gap in knowledge that initiated our current study. Using an automated and quantitative Wes assay (ProteinSimple, San Jose, CA), we show that ISGylation is significantly elevated in the lumbar spinal cords (SC-Ls), but not in the occipital lobes (OC-Ls), obtained from TBI-ALS compared with ALS veterans without a previous history of TBI (nonTBI-ALS). We also show that ISGylation is significantly elevated in lymphocyte cell lines (LCLs) generated from blood samples obtained from nonveteran ALS patients (n = 47) compared with normal LCLs (n = 44). On the basis of these observations, we propose that ISGylation may be a novel diagnostic biomarker for identifying ALS in TBI-exposed veterans. Moreover, since ISGylation is also elevated in ALS patients (veterans and nonveterans) compared with normal individuals, it may also be used as a biomarker for predicting ALS disease onset in general. Notably, our results that ISGylation is elevated in CSF samples of TBI-ALS veterans suggest that easily accessible CSF could be used to predict a risk for ALS in TBI-exposed veterans.

MATERIALS AND METHODS

Human SC-L and Occipital Lobe Tissues

De-identified SC-L, occipital tissues, and CSF samples from TBI-ALS, nonTBI-ALS, and normal veterans were provided by the Department of Veterans Affairs Biorepository (Boston, MA) VA Merit review BX002466 (Table 1). All tissues were obtained from male subjects except 1 female subject (#1 in Table 1). The RNA Integrity Number (RIN) values ranged from 5.7 to 6.2 for normal, 2.7 to 6.8 for nonTBI-ALS, and 4.7 to 7.5 for TBI-ALS samples, with median RINs of 5.9, 5.1, and 5.9, respectively. The postmortem interval (PMI) of autopsies ranged from 1.5 to 4.0 hours for normal, 1 to 8 hours for nonTBI-ALS, and 1 to 5 hours for TBI-ALS with median PMIs of 2.75 hours, 3.8 hours, and 2.3 hours, respectively. TBI and chronic traumatic encephalopathy (CTE) in subjects were determined as described in (15) (information provided by the VA Brain Bank). No patient contact was made; therefore, this study was exempt from the Institutional Review Board Committee at LSUHSC.

TABLE 1.

Tissues From Normal, TBI-ALS, and NonTBI ALS Veterans

ID #s	Primary Neuropathological Diagnosis	TBI	CTE	Tissue Type	Age
1	Normal			OC-L, SC-L	81
2	Normal			OC-L	62
3	Normal			OC-L	68
4	Normal			OC-L, SC-L	Unknown
5	Normal			OC-L, SC-L	Unknown
6	Normal			OC-L, SC-L	Unknown
7	ALS			OC-L, SC-L	73
8	ALS			SC-L, OC-L	71
9	ALS			SC-L	67
10	ALS			OC-L, SC-L	56
11	ALS			OC-L, SC-L	66
12	ALS			OC-L, SC-L	77
13	ALS			OC-L, SC-L	73
14	ALS			OC-L, SC-L	87
15	ALS			OC-L, SC-L	64
16	ALS			OC-L, SC-L	62
17	ALS			OC-L, SC-L	66
18	ALS			OC-L, SC-L	78
19	ALS			OC-L, SC-L	73
20	ALS		+	OC-L, SC-L	80
21	ALS			OC-L, SC-L	76
22	ALS			OC-L, SC-L	76
23	ALS			OC-L, SC-L	74
24	ALS			CSF	60
25	ALS			CSF	62
26	ALS			CSF	76
27	ALS	+	+	OC-L, SC-L	63
28	ALS	+		OC-L, SC-L, CSF	88
29	ALS	+		OC-L, SC-L	69
30	ALS	+	+	OC-L, SC-L	41
31	ALS	+	+	OC-L, SC-L	80
32	ALS	+		OC-L, SC-L, CSF	57
33	ALS	+	+	OC-L, SC-L, CSF	88
34	ALS	+	+	OC-L, SC-L	54
35	ALS	+		OC-L, SC-L, CSF	80
36	ALS	+	+*	OCL, SCL	79
37	ALS	+		OC-L, CSF	53
38	ALS	+		OC-L	73
39	ALS	+	+	OC-L, SC-L, CSF	55
40	ALS	+		OC-L	75

Information made available with human tissues from The Veterans Affairs Biorepository Brain Bank (Boston, MA) is tabulated.

OC-L, occipital lobes; SC-L, lumbar spinal cords; TBI, traumatic brain injury; ALS, amyotrophic lateral sclerosis; CTE, chronic traumatic encephalopathy; CSF, cerebral spinal fluid.

^*CTE and FTLD-TDP: This case had both CTE and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP).

Human LCLs

De-identified normal (n = 44) and ALS (n = 47) (harboring Chromosome 9 open reading frame 72 gene [C9orf72] mutation) LCLs were obtained from the Coriell Cell Repository for Medical Research (Camden, NJ). All cells were cultured in Roswell Park Memorial Institute Medium 1640 with 2 mM l-glutamine and 15% fetal bovine serum (without inactivation) and grown in a 37°C incubator with 5% CO₂. Normal and ALS patient-derived LCLs were chosen randomly regardless of age, race, and sex (Table 2).

TABLE 2.

Lymphocyte Cell Lines From Normal Individuals and ALS Patients

ALS Lymphocytes
	ID#	Description	Age at Sampling	Sex	Race	Gene	Mutation
1	ND07669	ALS 1	42 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
2	ND08957	ALS 1	48 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
3	ND09407	ALS 1	57 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
4	ND09204	ALS1	61 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
5	ND09373	ALS1	57 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
6	ND08544	undiagnosed	61 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
7	ND07489	ALS 1	68 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
8	ND09375	ALS1	57 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
9	ND06751	ALS1	52 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
10	ND08078	ALS1	54 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
11	ND06769	ALS 1	46 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
12	ND10000	ALS1	51 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
13	ND10023	ALS1	63 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
14	ND10101	ALS1	62 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
15	ND10284	ALS1	50 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
16	ND10808	ALS1	57 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
17	ND11081	ALS1	62 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
18	ND10973	ALS1	64 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
19	ND11411	ALS1	54 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
20	ND11494	ALS1	59 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
21	ND11252	ALS1	49 years	Male	Other	C9ORF72	(GGGGCC)n expansion
22	ND11548	ALS1	69 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
23	ND11917	ALS1	70 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
24	ND12089	ALS1	62 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
25	ND12099	ALS1	49 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
26	ND12100	ALS1	56 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
27	ND11933	ALS1	48 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
28	ND11680	ALS1	69 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
29	ND12161	ALS1	55 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
30	ND12199	ALS1	63 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
31	ND12200	ALS1	69 years	Female	Other	C9ORF72	(GGGGCC)n expansion
32	ND12277	ALS1	48 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
33	ND12455	ALS1	81 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
34	ND13939	ALS1	49 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
35	ND13682	ALS1	59 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
36	ND19454	ALS1	80 years	Male	Caucasian	Unknown	Unknown
37	ND14186	ALS1	65 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
38	ND14954	ALS1	61 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
39	ND13944	ALS1	49 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
40	ND13682	ALS1	59 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
41	ND14339	ALS1	75 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
42	ND13216	ALS1	57 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
43	ND19454	ALS1	80 years	Male	Caucasian	Unknown	Unknown
44	ND14186	ALS1	65 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
45	ND14954	ALS1	61 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion
46	ND12754	ALS1	70 years	Female	Caucasian	C9ORF72	(GGGGCC)n expansion
47	ND10773	ALS1	65 years	Male	Caucasian	C9ORF72	(GGGGCC)n expansion

Normal Lymphocytes
	ID #s	Description	Age at Sampling	Sex	Race
1	ND00700	Population Control	74 years	Female	Caucasian
2	ND00528	Population Control	75 years	Male	Caucasian
3	ND02405	Population Control	67 years	Female	Caucasian
4	ND03123	Population Control	68 years	Male	Caucasian
5	ND00940	Population Control	75 years	Male	Caucasian
6	ND01042	Population Control	71 years	Male	Caucasian
7	ND02645	Population Control	63 years	Female	Caucasian
8	ND03125	Population Control	74 years	Male	Caucasian
9	ND01693	Population Control	76 years	Female	Caucasian
10	ND02862	Population Control	75 years	Female	Caucasian
11	ND03628	Population Control	74 years	Male	Caucasian
12	ND03447	Population Control	84 years	Female	Caucasian
13	ND03661	Population Control	72 years	Female	Caucasian
14	ND03663	Population Control	73 years	Male	Caucasian
15	ND03627	Population Control	69 years	Male	Caucasian
16	ND04016	Population Control	65 years	Male	Caucasian
17	ND04017	Population Control	58 years	Male	Caucasian
18	ND04045	Population Control	64 years	Male	Caucasian
19	ND04240	Population Control	64 years	Female	Caucasian
20	ND04533	Population Control	72 years	Male	Caucasian
21	ND03970	Population Control	80 years	Male	Caucasian
22	ND05027	Population Control	61 years	Male	Caucasian
23	ND04531	Population Control	62 years	Male	Caucasian
24	ND04104	Population Control	69 years	Male	Caucasian
25	ND04903	Population Control	60 years	Female	Caucasian
26	ND05067	Population Control	55 years	Male	Caucasian
27	ND05283	Population Control	64 years	Male	Caucasian
28	ND05299	Population Control	58 years	Male	Caucasian
29	ND05369	Population Control	84 years	Male	Caucasian
30	ND05370	Population Control	55 years	Male	Caucasian
31	ND05372	Population Control	71 years	Female	Caucasian
32	ND05421	Population Control	59 years	Female	Caucasian
33	ND03836	Population Control	70 years	Female	Caucasian
34	ND03870	Population Control	64 years	Male	Caucasian
35	ND03746	Population Control	81 years	Female	Caucasian
36	ND03792	Population Control	57 years	Female	Caucasian
37	ND02864	Population Control	58 years	Female	Caucasian
37	ND03662	Population Control	78 years	Male	Caucasian
39	ND01519	Population Control	76 years	Male	Caucasian
40	ND03970	Population Control	80 years	Male	Caucasian
41	ND04276	Population Control	71 years	Male	Caucasian
42	ND04312	Population Control	55 years	Female	Caucasian
43	ND04586	Population Control	74 years	Female	Caucasian
44	ND05256	Population Control	66 years	Female	Caucasian

Information made available on the Coriell website is tabulated.

Human ISG15 Antibodies

Polyclonal antibodies were raised against purified hISG15 protein from Boston Biochem (Cambridge, MA) using custom antibody services provided by Cocalico Biologicals, Inc. (Reamstown, PA).

ISG15 Immunodetection by the Wes Assay

Human Tissue

Western analysis of ISGylation was performed using the Wes system from ProteinSimple (San Jose, CA) and a 12–230 kDa Separation Module (ProteinSimple, PN: SM-W004-1) following the instruction manual provided by the manufacturer. With the Wes system, all protein loading, separation, immunoblotting, washing, detection, and quantitative analysis of data are automated. All proteins are separated in capillaries as they migrate through a stacking and separation matrix. The separated proteins are immobilized to the capillary wall via proprietary photoactivated capture chemistry. Target proteins are then identified with a primary antibody (in our case, an ISG15-specific antibody), and subsequent immunodetection is carried out using a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent substrate (ProteinSimple reagents). Molecular weight and signal for immunodetected proteins are automatically recorded. Briefly, spinal cord tissues were lysed in 4% sodium dodecyl sulfate (SDS) lysis buffer (50 mM Tris-HCl, pH 7.5, 4% SDS) with phosphatase and protease inhibitors (10 mL per 1 mL of buffer), sonicated, and boiled for 10 minutes at 100°C. Lysates were clarified via centrifugation at 13 000 rpm for 2 minutes. Protein concentrations were determined by measuring absorbance at 280 nm using a Beckman Coulter Spectrophotometer and diluted to 0.01 μg/μl. Samples were then boiled for 10 minutes at 100°C before loading. Four parts of diluted samples were combined with 1 part of 5× Fluorescent Master Mix (containing 5× sample buffer, 5× fluorescent standard, and 200 mM DTT included in the ProteinSimple Standard Pack 1, PN: PS-ST01-8) and heated at 95°C for 5 minutes. The prepared samples, blocking reagent (antibody diluent), primary antibodies, HRP-conjugated secondary antibodies, and chemiluminescent substrate were dispensed into their designated wells on a Wes-25 size assay plate as suggested by the manufacturer. Antibodies used in the present study include anti-ISG15 primary (1:250 in Wes antibody buffer 2), antiβ-actin primary (1:500 in Wes antibody buffer 2) (Cambridge, MA) anti-ubiquitin primary (1:500 in Wes antibody buffer 2) (from Dr. Arthur Haas, LSUHSC, New Orleans, LA), and an HRP-conjugated rabbit and mouse secondary (ProteinSimple Detection Modules, PNs: DM-001 and DM-002, respectively). A biotinylated ladder included in the Standard Pack 1 provided molecular weight standards for the assay. The loaded plate was spun at 2500 rpm at room temperature for 5 minutes using an S6096 rotor in a Beckman Coulter Allegra X-30X centrifuge and then placed into the fully automated Wes machine along with an 8×25 capillary cartridge (included in the Separation Module). The company set Wes protocol was used and is as follows: Separation for 35 minutes at 375 V, antibody diluent for 5 minutes, primary antibody for 30 minutes, and secondary antibody for 30 minutes. After completion of the assay, automatic peak detection was assessed to determine if any manual corrections were needed for the standard peaks. Protein quantitation was performed using Compass Simple Western software.

CSF Samples

Four percent SDS lysis buffer (50 mM Tris-HCl, pH 7.5, 4% SDS) with phosphatase and protease inhibitors (10 mL per 1 mL of buffer) was added to the CSF samples, sonicated, and boiled for 10 minutes at 100°C. Lysates were clarified via centrifugation at 13 000 rpm for 2 minutes. Protein concentrations were determined by measuring absorbance at 280 nm using a Beckman Coulter Spectrophotometer and diluted to 0.01 μg/μl. Samples were boiled for 10 minutes at 100°C before loading, and immunodetection of ISG15 and ubiquitin conjugates was carried out using the Wes system as described above.

Lymphoblast Cells

Cells were lysed in 4% SDS lysis buffer (50 mM Tris-HCl, pH 7.5, 4% SDS), sonicated, and boiled for 10 minutes at 100°C. Lysates were clarified via centrifugation at 13 000 rpm for 2 minutes. Protein concentrations were determined by measuring absorbance at 280 nm using a Beckman Coulter Spectrophotometer, and proteins were adjusted to equal protein amounts. Immunodetection of ISG15 and β-actin was carried out using the Wes system as described above.

Statistical Analysis

Statistical analysis (unpaired t-test) was performed with GraphPad software. A p value < 0.05 was considered statistically significant.

RESULTS

Validation of Anti-ISG15 and Anti-ubiquitin Antibodies in the Wes Assay

ISG15, an anti-ubiquitin-cross reactive protein, is elevated in A-T cells (11, 16). To confirm that anti-ISG15 antibodies detect ISG15 and ISGylation, and not ubiquitin and ubiquitination, we assessed levels of free ISG15 and ISGylation in the cell lysates of A-T (overexpressing ISG15) and ISG15-silenced A-T cells (11) using the Wes as described in Materials and Methods. The same cell lysates containing equal protein were loaded in 2 sets and probed with anti-ISG15 (lanes 1 and 2) and anti-ubiquitin (lanes 3 and 4) antibodies. As presented in Figure 1, anti-ISG15 antibodies showed immunoreactivity with free ISG15 and ISGylation in ISG15 overexpressing A-T cells but not ISG15-silenced A-T cells in the Wes assay (Fig. 1, lanes 1 and 2). Alternatively, anti-ubiquitin antibody showed immunoreactivity with free ubiquitin and ubiquitin conjugates in both ISG15 overexpressing and ISG15-silenced A-T cells (lanes 3 and 4). ISG15 inhibits polyubiquitylation of cellular proteins. Consequently, the steady-state levels of polyubiquitylated proteins are decreased in ISG15 overexpressing A-T cells, while polyubiquitin levels are restored in ISG15-silenced A-T cells (10). Lower exposure of the Wes images confirmed these observations (right panel, compare lanes 3 and 4). These results thus validate the authenticity of anti-ISG15 and anti-ubiquitin antibodies to assess protein ISGylation and ubiquitination in the Wes assay.

FIGURE 1.

Validation of anti-ISG15 and anti-ubiquitin antibodies in the Wes assay. ISG15 (lanes 1 and 2) and ubiquitin (lanes 3 and 4) expression in A-T and ISG15-silenced A-T cell lysates were assessed on Wes using ISG15- and ubiquitin-specific antisera, respectively. High (left panel) and low (right panel) exposures of Wes image are shown. Double-headed arrow indicates a break in the original gel.

ISGylation Is Elevated in TBI-exposed ALS Veterans

Using anti-ISG15 antisera described above, we assessed ISGylation in SC-Ls obtained postmortem from TBI-ALS, nonTBI-ALS, and normal veterans using the Wes assay. As negative controls, we assessed ISGylation in OC-Ls (occipital brain tissues) obtained from the same TBI-ALS, nonTBI-ALS, and normal veterans. Several Wes blots were developed to assess ISGylation and free ISG15 in SC-L and OC-Ls described in Table 1. Representative blots generated by Wes are shown in Figure 2A (SC-Ls) and 2B (OC-Ls). Intensities of free ISG15, ISGylation (area under peaks spanning from 83 to 250 kDa comprising high molecular weight ISG15 conjugates), and β-actin were quantitated using Compass for Simple Western software. The box and whisker plot shows mean values of the ratio between ISGylation/β-actin (Fig. 2C, bars 4–6) and free ISG15/β-actin (Fig. 2D, bars 4–6). When normalized to β-actin, ISGylation levels were significantly increased in SC-Ls of TBI-ALS veterans (Fig. 2C, bar 6) compared with nonTBI-ALS (Fig. 2C, bar 5) (p = 0.0164) and normal veterans (Fig. 2C, bar 4) (ISGylation in normal veterans < nonTBI-ALS veterans < TBI-ALS veterans). Levels of free ISG15 were also elevated in SC-Ls of TBI-ALS compared with nonTBI-ALS and normal veterans (Fig. 2D, compare bars 5 and 6). However, the increase in free ISG15 levels was not as dramatic as ISGylation in TBI-ALS compared with nonTBI-ALS veterans and data did not reach statistical significance. In contrast to SC-Ls, ISGylation and free ISG15 remained the same in OC-Ls of all 3 groups (Fig. 2C, D, bars 1–3). These results suggest that ISGylation is predominantly increased in the SC-Ls of TBI-exposed ALS veterans, and data using OC-Ls suggest that elevated ISGylation is seen in ALS-disease-specific spinal cord tissues only. Furthermore, comparable RIN and PIM values (see Materials and Methods) suggest that elevated ISGylation is not due to differences in tissue integrity and PMI times.

FIGURE 2.

ISGylation is increased in the spinal cords of TBI-exposed ALS veterans. (A, B) ISGylation and free ISG15 levels in SC-Ls (A) and OC-Ls (B) were assessed using Wes (ProteinSimple) with ISG15-specific antisera as described in Materials and Methods. For loading controls, the same samples were probed using an antibody against β-actin. Data are shown in a gel view. (A) The first 3 lanes (control samples) were taken from a distinct Wes run and hence are shown separated from the remaining blot by a double-headed arrow. (C, D) Intensities of free ISG15, ISGylation (83–250 kDa region), and β-actin bands were quantitated using Compass for Simple Western software. The boxplot graphs show values of the ratio between ISGylatio/β-actin (C) and free ISG15/β-actin (D), measured from SC-Ls and OC-Ls. Error bars represent ± SEM.

We also assessed ISGylation in a few available CSF samples of TBI-ALS and nonTBI-ALS veterans. As shown in Figure 3, like SC-Ls, ISGylation levels (area under peaks spanning from 83 to 250 kDa) were increased in CSF samples of TBI-ALS veterans (left panel, lanes 4–9, and boxplot graph) compared with nonTBI-ALS (left panel, lanes 1–3, and boxplot graph). A previous study reported that ubiquitin levels reached a 4-fold increase in CSF samples in patients with TBI (17). Therefore, we probed the same set of samples with anti-ubiquitin antibodies (right panel). Similar to the literature report (17), CSF ubiquitin conjugates were increased 4-fold in TBI-ALS (right panel, lanes 4–9, and boxplot graph) compared with nonTBI ALS (right panel, lanes 1–3, and boxplot graph) veterans. These 2 independent studies (Fig. 3 and [17]) using distinct samples and showing a similar trend of increased ubiquitin in CSF samples from TBI-injured veterans warrants further investigation with a larger sample size of normal and patient-derived CSF samples to confirm these observations.

FIGURE 3.

ISGylation is increased in CSF samples of TBI-exposed ALS veterans. ISGylation levels in CSF samples were assessed using Wes with ISG15-specific antisera as described in Materials and Methods (left panel). The same CSF samples were loaded on the same cartridge and assessed with ubiquitin-specific antisera (right panel). Data are shown in a gel view. Double-headed arrow indicates a break in the original gel (a lane containing a misdiagnosed sample was removed from the original gel). ISGylated (left panel) and ubiquitinated (right panel) bands (83–250 kDa) were quantitated using Compass for Simple Western software and values are plotted in the boxplot graphs. Error bars represent ± SEM. See Table 1 for information on ID numbers.

Together, these results suggest that ISGylation and ubiquitination are increased in TBI-exposed ALS veterans, and could serve as a biomarker for TBI-ALS veterans.

ISGylation Is Elevated in ALS LCLs

In parallel, we assessed ISGylation in lymphocytes derived from normal (n = 44) and ALS patients with C9orf72 mutations (with C9orf72 repeat expansions) (n = 47; Table 2). A representative blot generated by the Wes machine is shown in Figure 4A. Intensities of free ISG15, ISGylation, and β-actin were quantitated as described above. The box and whisker plot shows the mean values of the ratio between free ISG15/β-actin (Fig. 4B) and ISGylation/β-actin (Fig. 4C). As shown in Figure 4B, ISGylation levels were significantly increased in lymphocytes obtained from ALS (p = 0.02) compared with normal individuals. No changes were noted in free ISG15 in ALS compared with normal lymphocytes.

FIGURE 4.

ISGylation is increased in lymphocyte cell lines (LCLs) derived from ALS patients. (A) Cell lysates were prepared and analyzed by Wes using an antihuman ISG15 antibody as described in Materials and Methods. For loading controls, the same samples were probed using an antibody against β-actin. (B, C) Intensities of free ISG15, ISGylation (83–250 kDa), and β-actin bands were quantitated as described in Figure 2. The boxplot graphs show values of the ratio between free ISG15/β-actin (B) and ISGylation/β-actin (C), measured from LCLs derived from ALS patients and normal individuals. Error bars represent ± SEM.

Together, our results highlight the importance of ISGylation as a diagnostic biomarker for identifying ALS in TBI-exposed veterans. Since ISGylation is also elevated in nonTBI-ALS veterans and nonveteran ALS patients compared with normal individuals, it may also be used as a biomarker for predicting ALS disease onset in general. Furthermore, our results that ISGylation is increased in CSF and LCLs suggest that CSF- and LCL-ISGylation can serve as a surrogate biomarker for ISGylation in the spinal cords of TBI-ALS veterans, nonTBI-ALS veterans, and nonveteran ALS patients.

DISCUSSION

Mounting evidence suggests that veterans who have served in the military are at a nearly 60% greater risk of being diagnosed with ALS than those with no history of military service (2). However, the cause of disease in normal individuals, and why the incidence of ALS is higher among veterans, is not known. Literature reports suggest that a history of TBI may be a risk factor for ALS in veterans (3–5). Unfortunately, the causal biochemical link between TBI and ALS remains unclear, rendering the development of effective treatments a difficult task. Moreover, with no effective diagnostic biomarkers available for predicting ALS in TBI-exposed veterans, therapies are less likely to be implemented in the early stages of its development when they are expected to have their most significant impact. Hence, both identification of diagnostic biomarker(s) that can predict the risk of ALS in TBI-exposed veterans and knowledge of the biochemical mechanism underlying the high incidence of ALS in these veterans are urgently needed. Since ISG15 is elevated in human ALS patients and mice (6), and in response to TBI exposure in mice, we speculated that TBI-mediated induction of ISGylation, an antagonist of the ubiquitin pathway (10, 11), induces proteinopathy, and consequently ALS, in TBI-exposed veterans. The current study was initiated to test whether ISGylation is indeed induced in TBI-exposed veterans diagnosed with ALS.

We chose to use a Wes system for assessing ISGylation, as this technique allowed us to detect and quantitate both free ISG15 and ISGylation (ISG15 conjugates) in the same cell lysates, which is not possible using other techniques like dot-blotting, immunohistostaining, or gene expression analyses. Moreover, unlike traditional Western blotting analysis, in the Wes system, all steps of Western blotting, including protein loading, separation, immunoblotting, washing, detection and quantitative analysis of data are automated. Thus, manual factors that can negatively impact reproducibility, quantitation, time to result, and overall reliability of the generated data are eliminated. Using the Wes system, our results have revealed that ISGylation is significantly elevated in TBI-exposed veterans diagnosed with ALS compared with veterans with no previous history of TBI but diagnosed with ALS and normal veterans (no apparent disease conditions). We do not know why ISGylation is elevated in response to TBI, and how ISGylation is elevated in the spinal cord and lymphocytes of ALS and TBI-exposed ALS veterans. However, literature reports indicate that TBI induces the expression of microRNA155 (miR155), which in turn induces Type 1 IFN, a primary regulator of ISG15 and its conjugating enzymes (18). Moreover, miR155 is upregulated in the spinal cords of end-stage ALS model (SOD1^G93A) mice (19). It is possible that IFN secreted by brains in response to TBI travel through CSF and blood, inducing ISGylation in spinal cords and lymphocytes, respectively. Studies are underway to test this hypothesis in our laboratory.

CTE is a neurodegenerative disease associated with repetitive mild TBI as well as potentially subconcussive impacts that typically occur in the context of contact sports (20), but may also occur during military service (15). CTE is diagnosed neuropathologically by the accumulation of hyperphosphorylated and aggregated tau in neurons, astrocytes, cell processes around small vessels and the depths of the cerebral sulci. In the current study, we have demonstrated that ISGylation is increased in SCLs obtained from ALS-TBI veterans compared with ALS veterans. Notably, 7 of 10 ALS-TBI veterans and 1 of 17 ALS veterans whose SCLs were analyzed for ISGylation in the current study showed evidence of CTE. However, due to the small sample size (TBI [n = 3] vs TBI-CTE [n = 7]), we do not know if the increase in ISGylation would be predictive of CTE in ALS veterans. Further studies with a larger sample size are needed to confirm if increased ISGylation is associated with CTE in TBI-exposed ALS veterans. In the current study, we have also demonstrated that ISGylation is increased in lymphocytes derived from ALS patients harboring C9orf72 mutations compared with normal subjects. Mutations in C9orf72 are the most commonly known genetic cause of ALS and are seen in ∼40% of patients with a family history and ∼10% of those without (21). Other than C9orf72, mutations in genes encoding copper-zinc superoxide dismutase (SOD1), transactive response DNA-binding protein 43 (TDP-43), and fused in sarcoma (FUS) account for >50% of the familial ALS cases (21). Whether ISGylation is elevated in lymphocytes/SC-Ls obtained from patients harboring these other mutations, is not known. In the current study, we also report that ubiquitin conjugates are increased in the CSF samples of TBI-ALS compared with nonTBI-ALS veterans. These results, although not statistically significant (most likely due to low sample size), agree with the previous literature report wherein authors have reported that ubiquitin levels are increased in CSF of TBI-exposed patients (17). Thus, our study and this independent literature study using distinct patient samples highlights the importance of ubiquitin/ISG15 conjugates as a biomarker for TBI-exposed civilians and veterans. The reason underlying increased ubiquitin (and ISG15) in CSF is not clear. However, neuronal cell damage has been suggested as an underlying cause of increased ubiquitin levels upon TBI in patients (17). Our findings of increased ubiquitin/ISG15 protein conjugate levels strongly suggest this possibility. In summary, in the current study we have demonstrated that ISGylation levels are higher in SC-Ls and CSFs of TBI-exposed ALS and nonTBI-ALS compared with normal veterans. Moreover, ISGylation levels are elevated in LCLs of ALS patients compared with normal patients. Together, these results suggest that ISGylation may be a potential diagnostic biomarker for predicting TBI (and possibly CTE) in ALS veterans and nonveteran ALS patients. Notably, TBI-induced CTE is common in athletes/football players (20). Moreover, ISG15 has been identified as a biomarker for neuronal injury (14), and neuronal injury is common to all neurodegenerative diseases. Other studies from our laboratory have revealed that ISGylation is also increased in ataxia telangiectasia, Parkinson, and Alzheimer neurodegenerative diseases. Thus, it appears that elevated ISGylation is a common trait of neurodegenerative diseases. However, the reliability of using ISGylation as a biomarker for diagnosing neurological disorders and TBI/CTE in football players requires further study.

Western-based diagnostic tests are currently licensed for use in veterinary and human clinical practices (22), rendering the Wes assay to measure ISGylation in fresh lymphocytes, and perhaps patient-derived CSF, a feasible approach. It is important to note that ISGylation is also elevated in most cancer and pathogen-infected cells. Therefore, this ISGylation test should be used in conjunction with physical phenotypes and patient medical history for accurate clinical diagnosis of ALS. Another blood-based biomarker test, the Alpha-Fetoprotein (AFP) test, utilizes this comprehensive approach since, like ISGylation, AFP is elevated in both cancer and recessive ataxias (e.g. ataxia telangiectasia). Admittedly, to translate the current data into a clinical biomarker, determination of factors such as a cutoff point for maximal sensitivity/specificity, and the rates of false negativity/positivity using large sample size, is required. The current study will form a foundation of such a larger biomarker study. Nevertheless, at present, no clinical tests are available that can assess the risk of ALS in TBI-exposed veterans, thereby making this biomarker study a novel one.

LIFT² and NIH/NINDS R21NS060960 grants support this work.