Leucine-rich α2-glycoprotein is a novel biomarker of neurodegenerative disease in human cerebrospinal fluid and causes neurodegeneration in mouse cerebral cortex

Abstract

Leucine-rich α2-glycoprotein (LRG) is a protein induced by inflammation. It contains a leucine-rich repeat (LRR) structure and easily binds with other molecules. However, the function of LRG in the brain during aging and neurodegenerative diseases has not been investigated. Here, we measured human LRG (hLRG) concentration in the cerebrospinal fluid (CSF) and observed hLRG expression in post-mortem human cerebral cortex. We then generated transgenic (Tg) mice that over-expressed mouse LRG (mLRG) in the brain to examine the effects of mLRG accumulation. Finally, we examined protein-protein interactions using a protein microarray method to screen proteins with a high affinity for hLRG. The CSF concentration of hLRG increases with age and is significantly higher in patients with Parkinson's disease with dementia (PDD) and progressive supranuclear palsy (PSP) than in healthy elderly people, idiopathic normal pressure hydrocephalus (iNPH) patients, and individuals with Alzheimer's disease (AD). Tg mice exhibited neuronal degeneration and neuronal decline. Accumulation of LRG in the brains of PDD and PSP patients is not a primary etiological factor, but it is thought to be one of the causes of neurodegeneration. It is anticipated that hLRG CSF levels will be a useful biomarker for the early diagnosis of PDD and PSP.

Figure 1. Human CSF analysis.

Relationships between age and CSF total protein and LRG levels. (A) CSF total protein level did not correlate with age (r = 0.031, p = 0.731) (B) CSF LRG level significantly correlated with age (r = 0.314, p < 0.0001) using Spearman rank correlation. (C) CSF LRG level reverse correlated with the MMSE score (r = -0.271, p = 0.003) using Spearman rank correlation. LRG levels were significantly higher in the MMSE ≤ 23 group (113.6 ± 70.0 ng/ml) than in the MMSE > 23 group (92.0 ± 58.3 ng/ml), Mann-Whitney U test, p < 0.05). (D) CSF LRG levels tended to be higher in the iNPH groups (106.0 ± 46.7 ng/ml) vs. NC groups (44.0 ± 25.2 ng/ml), Mann-Whitney U test, p < 0.001. Concentrations of LRG in the PDD/DLB (251.5 ± 106.5 ng/ml) and PSP (261.1 ± 182.9 ng/ml) groups were significantly higher than those in the NC, iNPH, and AD groups (95.1 ± 64.4 ng/ml), Mann-Whitney U test, p < 0.001; PD/DLB vs. NC, p < 0.001; PSP vs. NC, p = 0.001; PDD/DLB vs. iNPH, p = 0.021, PSP vs. iNPH; p < 0.001, PDD/DLB vs. AD; p = 0.013, PSP vs. AD). ROC analysis of CSF biomarkers. LRG was the best discriminating biomarker for (E) PDD/DLB patients vs. NC, (F) PSP patients vs. NC, (G) iNPH patients vs. NC, (H) iNPH patients vs. PDD/DLB, and (I) iNPH patients vs. PSP, LRG was the most discriminating biomarker. CSF = cerebrospinal fluid, LRG = leucine-rich α2-glycoprotein. MMSE = Mini-Mental State Examination, iNPH = idiopathic normal pressure hydrocephalus, NC = normal control, PDD = Parkinson disease with dementia, DLB = dementia with Lewy bodies, PSP = Progressive Supranuclear Palsy, AD = Alzheimer disease, Amyloid(1-42) = amyloid beta peptide 1-42, p-TAU = phosphorylated tau, TAU = total tau.

Figure 2. LRG immunostaining of autopsied brains.

(A) Western blotting of hLRG-transfected COS cell lysates showed strong expression of a 38-kDa band corresponding to LRG (arrow). a: COS cell lysate, b: hLRG-transfected COS cell lysates. (B) Immunoreactivity in hLRG-transfected cells was confirmed by immunostaining with anti-hLRG(329) rabbit IgG. COS cells, CHO-K1 cells, HEK293 cells. Scale bar = 20µm. (C) hLRG immunoreactivity in some glial cells and neurons in the internal pyramidal layer and multiform layer of the frontal cortex. Compared to the NC, LRG-immunoreactive cells were increased in iNPH. There was a significant increase in LRG-positive cells in PDD and PSP. With AD, we confirmed strong LRG immunoreactivity in neurons rather than in glial cells. Scale bar = 20µm (NC, iNPH, PDD, PSP and AD) and 10µm (Neuron and Glial cell). NC: 74-year-old male, frontal cortex iNPH: 76-year-old, male, frontal cortex. PDD: 69-year-old, male, frontal cortex. PSP: 65-year-old, male, temporal cortex, glial cell. AD: 82-year-old, male, frontal cortex, neuron. (D) Quantitation of hLRG immunoreactive cells in the cerebral cortex. Stained cells in a 0.05-mm² area of each cortical region were counted by independently by two investigators. The values on the y-axis represent the number of immunoreactive cells and are expressed as the means ± standard deviations for five areas in each case, and the results were analyzed using a one-way ANOVA followed by Dunnett’s test, *p < 0.05, **p < 0.001. Bars, + SD.

Figure 3. Age-related changes in mouse cerebral cortex.

(A) Western blot analysis. Compared to the 4- and 8-week-old mice, LRG protein levels were higher in 48-week-old mice. (B) The mean mLRG intensity in the 48-week-old mice was significantly increased compared with that in the 4- and 8-week-old mice as determined by one-way ANOVA followed by Dunnett’s test, **p < 0.001, Bars + SD. (C) Only weak LRG immunoreactivity was observed in the neurons and glial cells of 4- and 8-week-old mice, whereas significant LRG immunoreactivity was found in the neurons and glial cells of 48- and 108-week-old mice (I). Scale bar = 10µm. (D) Macroscopic findings of brain from 8-week-old mice. Scale bar = 5 mm. (E) Hematoxylin and eosin (HE) and Kluver-Barrera (KB) stains of coronal sections from 8-week-old mice and (F) 48-week-old mice. Scale bar = 500µm. (G) LRG proteins were significantly higher in Tg mice than in WT. (H) Densitometry analysis shows that LRG levels are approximately 2-to-4-fold higher in Tg mice, as determined by Mann-Whitney U test; *p < 0.05, **p < 0.001, Bars, + SD. (I) The cerebral cortex of 8-week-old and 48-week-old Tg mice presented significantly greater LRG immunoreactivity in the cytoplasm of neurons and glial cells than that of WT mice at 8 weeks and 48 weeks. Scale bar = 10µm. (J) Cerebral cortex of the 8- and 48-week-old Tg mice exhibited more LRG mRNA in the cytoplasm of neurons and glial cells than that of 8- and 48-week-old WT mice. Each red dot indicates one mRNA copy. Scale bar = 10µm. WT = wild type, Tg = transgenic.

Figure 4. Immunostaining results for LRG transgenic mice.

(A) Immunostaining of the cerebral cortex of 4-, 8-, and 48-week old mice using NeuN antibody. 4-week-old WT mice, 4-week-old Tg mice, 8-week-old WT mice, 8-week-old Tg mice, 48-week-old WT mice, and 48-week-old Tg mice. Scale bar = 20µm. (B) A difference in the number of NeuN-positive cells was already observed between 4-week-old Tg and WT mice, but with 8- and 48-week old mice, there was a significant decline in the number of NeuN-positive cells in Tg mice compared to that in WT as determined by Mann-Whitney U test, **p < 0.001, Bars, + SD. (C) Cortical immunostaining for phosphorylated tau in 8-week-old WT, 8-week-old Tg, Tg mice neurons (lower panel, arrow), and Tg mice glial cells (lower panel, arrow head). Scale bar = 20µm (upper panel) and 10µm (lower panel). (D) Tg cerebral cortex demonstrated significantly more neurons and glial cells that were positive for phosphorylated tau than WT as determined by Mann-Whitney U test, **p < 0.001, Bars + SD. (E) NF-L immunostaining in the cortex of 8-week-old WT mice and the cortex of 8-week-old Tg mice. Large, winding dendritic neurons in Tg mice suggested that neurodegeneration had occurred. Scale bar = 20µm (upper panel) and 10µm (lower panel). WT = wild type, Tg = transgenic.