Chitotriosidase, a biomarker of amyotrophic lateral sclerosis, accentuates neurodegeneration in spinal motor neurons through neuroinflammation

Abstract

Background: Cerebrospinal fluid from amyotrophic lateral sclerosis patients (ALS-CSF) induces neurodegenerative changes in motor neurons and gliosis in sporadic ALS models. Search for identification of toxic factor(s) in CSF revealed an enhancement in the level and enzyme activity of chitotriosidase (CHIT-1). Here, we have investigated its upregulation in a large cohort of samples and more importantly its role in ALS pathogenesis in a rat model.

Methods: CHIT-1 level in CSF samples from ALS (n = 158), non-ALS (n = 12) and normal (n = 48) subjects were measured using ELISA. Enzyme activity was also assessed (ALS, n = 56; non-ALS, n = 10 and normal-CSF, n = 45). Recombinant CHIT-1 was intrathecally injected into Wistar rat neonates. Lumbar spinal cord sections were stained for Iba1, glial fibrillary acidic protein and choline acetyl transferase to identify microglia, astrocytes and motor neurons respectively after 48 h of injection. Levels of tumour necrosis factor-α and interleukin-6 were measured by ELISA.

Findings: CHIT-1 level in ALS-CSF samples was increased by 20-fold and it can distinguish ALS patients with a sensitivity of 87% and specificity of 83.3% at a cut off level of 1405.43 pg/ml. Enzyme activity of CHIT-1 was also 15-fold higher in ALS-CSF and has a sensitivity of 80.4% and specificity of 80% at cut off value of 0.1077989 μmol/μl/min. Combining CHIT-1 level and activity together gave a positive predictive value of 97.78% and negative predictive value of 100%. Administration of CHIT-1 increased microglial numbers and astrogliosis in the ventral horn with a concomitant increase in the levels of pro-inflammatory cytokines. Amoeboid-shaped microglial and astroglial cells were also present around the central canal. CHIT-1 administration also resulted in the reduction of motor neurons.

Conclusions: CHIT-1, an early diagnostic biomarker of sporadic ALS, activates glia priming them to attain a toxic phenotype resulting in neuroinflammation leading to motor neuronal death.

Keywords: Amyotrophic lateral sclerosis; Biomarker; Chitotriosidase; Glia; Neurodegeneration; Neuroinflammation.

Fig. 1

CHIT-1 levels in CSF of ALS patients and controls. ELISA showed increased levels of CHIT-1 compared to N-CSF and NALS-CSF (****p < 0.0001 vs. N-CSF and ^$$p < 0.01 vs NALS-CSF) (a). Receiver operating curve (ROC) analysis (b). Increased levels of CHIT-1 in CSF of patients with lesser disease duration (****p < 0.0001 N-CSF vs. ALS-CSF up to 12 months, *p < 0.05 N-CSF vs. ALS-CSF > 12 months and **p < 0.01 ALS-CSF up to 12 months vs. ALS-CSF > 12 months (c). Correlation between CHIT-1 levels and disease duration (d). Increased level of CHIT-1 in definite cases compared to probable/possible cases of ALS (**p < 0.01 probable/possible vs. definite) (e). No significant difference was noticed between male vs. female (f) and bulbar vs. limb onset (g) of ALS. Correlation between CHIT-1 levels and ALS-FRS score (h)

Fig. 2

CHIT-1 enzyme activity in ALS-CSF and control CSF. Increased CHIT-1 activity in ALS-CSF compared to N-CSF and NALS-CSF (***p < 0.001 vs. N-CSF and ^$$p < 0.01 vs. NALS-CSF) (a). Receiver operating curve (ROC) analysis (b)

Fig. 3

Effect of in vivo administration of CHIT-1 in glia. Representative confocal micrographs (merged) of spinal cord sections co-labeled with Iba1 (green) and GFAP (red) in normal control (NC; a), Buffer (b), CHIT-1 (c–f) and positive control (ALS, g) groups. Note the increase in Iba1 labelled microglia in CHIT-1 (mainly in C100) and ALS groups. An increase in GFAP expression was also observed in CHIT-1 (c–f) and ALS (g) groups. Note the change in morphology of the Iba1 labelled microglia from long process bearing ones in control (h) to those bearing short processes in CHIT-1 group (i). Scale bar = 150 μm (for all images). CHIT-1 induces a significant upregulation of Iba1 positive microglial cells at a dose of 100 pg similar to ALS-CSF group unlike buffer and normal controls (*p < 0.05 C100 and ALS vs. NC; ^$p < 0.05 C100 and ALS vs. buffer) (j). Histogram representing enhanced expression of GFAP in ventral horn white matter in CHIT-1 group compared to buffer and normal controls in terms of intensity (**p < 0.01 C50, C100, C200 vs. NC; ***p < 0.001 C500, ALS vs. NC and ^$$p < 0.01 C50, C100, C200 vs. buffer; ^$$$p < 0.001 C500 and ALS vs. buffer) (k) and area (*p < 0.05 C50 vs. NC; **p < 0.01 C100, C200, C500, ALS vs. NC and ^$$p < 0.01 NC vs. C50, C100, C200, C500 and ALS) (l)

Fig. 4

Effect of CHIT-1 on glia in different regions of the spinal cord. Representative confocal micrographs showing Iba1 (green) and GFAP (red) expression in the spinal cord sections (a–f). Significant amount of gliosis was noted in the ventral horn (V) compared to dorsal horn (D). The yellow coloured line demarcates the areas (b). The higher magnification images show the presence of astrocytes (red, small arrows, (e)) in the grey matter and also the Iba1 (green, big arrows, (f)) immunoreactive microglia surrounding the motor neurons (*). Scale bars are indicated. Representative confocal photomicrographs showing Iba1 (green) and GFAP (red) expression around the central canal (arrows) (g–o). Note the faint staining in normal control (NC, (g, h, i)) whereas both the markers show upregulation in the 50 and 100 pg concentrations of CHIT-1 (C50 and C100) (j–o). Note the presence of amoeboid shaped microglia within the vicinity of the canal (arrowheads) (o). Scale bars are indicated

Fig. 5

Effect of CHIT-1 on proinflammatory molecules. Histogram representing enhanced level of TNFα in lumbar spinal cord lysates of CHIT-1 (500 pg) and ALS group and compared to buffer control (*p < 0.05 C500, ALS vs. buffer) (a). Increased level of IL6 in lumbar spinal cord lysates of CHIT-1 (500 pg) and ALS group and compared to buffer control (*p < 0.05 C500 vs. buffer; **p < 0.01 ALS vs. buffer) (b)

Fig. 6

Effect of CHIT-1 on motor neurons and axonal density. Representative confocal images (merged) of spinal cord sections co-labeled for ChAT (green) and SMI-31 (red) in control [buffer, (a)], CHIT-1 [C50, 100, 200, 500 pg; (b–e)] and also ALS-CSF injected (f) groups. Scale bar = 150 μm (for all images). CHIT-1 induces loss of ChAT positive motor neuronal cells, similar to ALS-CSF, at a dose of 500 pg. (**p < 0.01 C500 vs. NC; ***p < 0.001 ALS vs. NC; ^$$p < 0.01 C500 and ALS vs. buffer) (g). Histogram representing area of phosphorylated neurofilaments (SMI-31) in white matter of ventral horn of spinal cord (h). Representative images of Nissl-stained spinal cord sections of normal control [NC, (i)], Buffer (j), CHIT-1 [C50, C100, C200, C500 pg; (k–n)] and ALS-CSF (o) injected groups. CHIT-1 (500 pg) and ALS-CSF results in motor neuronal loss (*p < 0.05 C500 vs NC; ***p < 0.001 ALS vs. NC; ^$$p < 0.01 C500 vs buffer; ^$$$p < 0.001 ALS vs. buffer) (p)

Fig. 7

Hypothesis of mechanisms of CHIT-1 induced neurotoxicity in ALS. CHIT-1 activates microglial cells by possibly interacting with receptors (e.g. transforming growth factor beta (TGF-β) or toll-like receptors) leading to the release of proinflammatory mediators. This leads to the heightened glial response with an increase in reactive astrocytes and activated microglia. Neurotoxic cytokines like IL-6, TNF-α along with ROS and nitric oxide released by both these glial cell types result in motor neuronal degeneration