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

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.

Small molecule modulator of aggrephagy regulates neuroinflammation to curb pathogenesis of neurodegeneration.

BACKGROUND: Plethora of efforts fails to yield a single drug to reverse the pathogenesis of Parkinson's disease (PD) and related α-synucleopathies. METHODS: Using chemical biology, we identified a small molecule inhibitor of c-abl kinase, PD180970 that could potentially clear the toxic protein aggregates. Genetic, molecular, cell biological and immunological assays were performed to understand the mechanism of action. In vivo preclinical disease model of PD was used to assess its neuroprotection efficacy. FINDINGS: In this report, we show the ability of a small molecule inhibitor of tyrosine kinases, PD180970, to induce autophagy (cell lines and mice midbrain) in an mTOR-independent manner and ameliorate the α-synuclein mediated toxicity. PD180970 also exerts anti-neuroinflammatory potential by inhibiting the release of proinflammatory cytokines such as IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1) through reduction of TLR-4 (toll like receptor-4) mediated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation. In vivo studies show that PD180970 is neuroprotective by degrading the toxic protein oligomers through induction of autophagy and subsiding the microglial activation. INTERPRETATION: These protective mechanisms ensure the negation of Parkinson's disease related motor impairments. FUND: This work was supported by Wellcome Trust/DBT India Alliance Intermediate Fellowship (500159-Z-09-Z), DST-SERB grant (EMR/2015/001946), DBT (BT/INF/22/SP27679/2018) and JNCASR intramural funds to RM, and SERB, DST (SR/SO/HS/0121/2012) to PAA, and DST-SERB (SB/YS/LS-215/2013) to JPC and BIRAC funding to ETA C-CAMP.

Sporadic amyotrophic lateral sclerosis (SALS) - skeletal muscle response to cerebrospinal fluid from SALS patients in a rat model.

Skeletal muscle atrophy is the most prominent feature of amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease of motor neurons. However, the contribution of skeletal muscle to disease progression remains elusive. Our previous studies have shown that intrathecal injection of cerebrospinal fluid from sporadic ALS patients (ALS-CSF) induces several degenerative changes in motor neurons and glia of neonatal rats. Here, we describe various pathologic events in the rat extensor digitorum longus muscle following intrathecal injection of ALS-CSF. Adenosine triphosphatase staining and electron microscopic (EM) analysis revealed significant atrophy and grouping of type 2 fibres in ALS-CSF-injected rats. Profound neuromuscular junction (NMJ) damage, such as fragmentation accompanied by denervation, were revealed by α-bungarotoxin immunostaining. Altered expression of key NMJ proteins, rapsyn and calpain, was also observed by immunoblotting. In addition, EM analysis showed sarcolemmal folding, Z-line streaming, structural alterations of mitochondria and dilated sarcoplasmic reticulum. The expression of trophic factors was affected, with significant downregulation of vascular endothelial growth factor (VEGF), marginal reduction in insulin-like growth factor-1 (IGF-1), and upregulation of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF). However, motor neurons might be unable to harness the enhanced levels of BDNF and GDNF, owing to impaired NMJs. We propose that ALS-CSF triggers motor neuronal degeneration, resulting in pathological changes in the skeletal muscle. Muscle damage further aggravates the motor neuronal pathology, because of the interdependency between them. This sets in a vicious cycle, leading to rapid and progressive loss of motor neurons, which could explain the relentless course of ALS.This article has an associated First Person interview with the first author of the paper.

VEGF alleviates ALS-CSF induced cytoplasmic accumulations of TDP-43 and FUS/TLS in NSC-34 cells.

Cytoplasmic mislocalisation and aggregation of TDP-43 and FUS/TLS proteins in spinal motor neurons contribute to the pathogenesis of the highly fatal disorder amyotrophic lateral sclerosis (ALS). We investigated the neuroprotective effect of VEGF on expression of these proteins in the motor neuronal cell line NSC-34 modelled to reminisce sporadic form of ALS. We studied the expression of TDP-43 and FUS/TLS proteins after exposure to ALS-CSF and following VEGF supplementation by quantitative confocal microscopy and electron microscopy. ALS-CSF caused cytoplasmic overexpression of both the proteins and stress-granule formation in the cells. These alterations were alleviated by VEGF supplementation. The related ultrastructural changes like nuclear membrane dysmorphism and p-bodies associated changes were also reversed. However the protein expression did not completely translocate to the nucleus, as some cells continued to show to cytoplasmic mislocalisation. Thus, the present findings indicate that VEGF alleviates TDP43 and FUS pathology by complimenting its role in controlling apoptosis and reversing choline acetyl transferase expression. Hence, VEGF appears to target multiple pathogenic processes in the neurodegenerative cascade of ALS.

Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients Induces Mitochondrial and Lysosomal Dysfunction.

In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS.

Role of VEGF and VEGFR2 Receptor in Reversal of ALS-CSF Induced Degeneration of NSC-34 Motor Neuron Cell Line.

Vascular endothelial growth factor (VEGF), the well-known angiogenic factor is both neurotrophic and neuroprotective. Altered VEGF signalling is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal degenerative disease of motor neurons. We have shown earlier that VEGF protects NSC-34 motor neuronal cell line, when exposed to cerebrospinal fluid (CSF) from sporadic ALS patients (ALS-CSF). Here, we have investigated the consequences of ALS-CSF and VEGF supplementation on the VEGFR2 receptor and endogenous VEGF expression. ALS-CSF caused significant down-regulation of VEGFR2 as well as the Calbindin-D28K levels, but not endogenous VEGF. Exogenous supplementation restored the depletion of VEGFR2 and Calbindin-D28K with a concomitant up-regulation of endogenous VEGF. The up-regulated caspase 3 in the ALS-CSF group was reinstated to basal levels along with a significant reduction in the number of TUNEL-positive cells. Electron photomicrographs of ALS-CSF-exposed cells divulged presence of cytoplasmic vacuoles alongside severe damage to organelles like mitochondria, endoplasmic reticulum, etc. Substantial recovery of most of the damaged organelles was noted in response to VEGF supplementation. While the enhancement in endogenous VEGF levels highlights the autocrine functions, the up-regulation of VEGFR2 receptor emphasizes the paracrine functions of VEGF in modulating its neuroprotective effect against ALS-CSF. The revival of cellular organellar structure, increased calbindin expression and enhanced survival in response to VEGF supplementation consolidates the opinion that VEGF indeed has a therapeutic potential in sporadic ALS.

Vascular endothelial growth factor attenuates neurodegenerative changes in the NSC-34 motor neuron cell line induced by cerebrospinal fluid of sporadic amyotrophic lateral sclerosis patients.

BACKGROUND: Motor neuron disease or amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motor neurons in the spinal cord as well as motor cortex. Recently, vascular endothelial growth factor (VEGF) has been identified as a neurotrophic factor in animal models of familial ALS and other neurological diseases. OBJECTIVE: The present study was designed to investigate the neuroprotective role of VEGF in the more prevalent sporadic form of ALS. METHODS: We studied the effect of VEGF on the NSC-34 cell line exposed to cerebrospinal fluid (CSF) from sporadic ALS patients (ALS-CSF) in terms of lactate dehydrogenase (LDH) assay as well as choline acetyltransferase (ChAT) and phosphorylated neurofilament expression by immunocytochemistry and confocal microscopy. NSC-34 cells were exposed to CSF from patients with definite ALS and compared to controls. LDH activity was assessed in the growth media, prior to and 24 h after the addition of VEGF to the cells. At similar time points, the cells were fixed and processed for immunocytochemistry to evaluate ChAT and phosphorylated neurofilament expression. RESULTS: Exposure to ALS-CSF caused morphological changes of NSC-34 cells like reduced differentiation and aggregation of phosphorylated neurofilaments. Enhanced LDH activity and reduced ChAT immunoreactivity were also observed. Addition of VEGF to NSC-34 cells exposed to ALS-CSF was protective in terms of reduced LDH activity and restoration of ChAT expression. CONCLUSION: The present study confirms that VEGF exerts a neuroprotective effect on the NSC-34 cell line by attenuating the degenerative changes induced by ALS-CSF. It thus has therapeutic potential in sporadic ALS.

Expression of GDNF receptors GFRalpha1 and RET is preserved in substantia nigra pars compacta of aging Asian Indians.

Glial derived neurotrophic factor (GDNF) protects dopaminergic nigral neurons and may prevent the progression of age-related motor deficits and Parkinson's disease. The multi-component receptor complex which mediates the neuroprotective action of GDNF comprises of GDNF receptor alpha1 (GFRalpha1), a ligand binding cell surface component and RET receptor tyrosine kinase (RET) the signaling component. The expression of both these receptors in the normally aging human substantia nigra pars compacta (SNpc) needs to be studied since GDNF infusion is being considered for restoration of the lost nigrostriatal function. In the present study, we used unbiased stereology to quantify the number of GFRalpha1 and RET immunoreactive neurons in human SNpc from 28 weeks of gestation to 88 years (n=31). We further determined the levels of immunostaining intensity using densitometric image analysis to measure changes in levels of receptor expression. Here we report that human nigral dopaminergic neurons express GFRalpha1 and RET receptors at all ages. There was no reduction in the number of neurons expressing these receptors as a function of age. Moreover, there was no age-related decline in immunostaining intensity of both these receptors. It is likely that preservation of GDNF receptors in the nigral neurons is because these receptors are constitutively expressed in the human SNpc and thus it is GDNF responsive thru aging. The sustained receptor protein expression could also be another marker of preserved nigrostriatal function in Asian Indians. The latter possibility explains our earlier observation that the melanized nigral neurons are preserved with age in the Asian Indians.

Cerebrospinal fluid from sporadic amyotrophic lateral sclerosis patients induces degeneration of a cultured motor neuron cell line.

We investigated the effect of Cerebrospinal Fluid (CSF) from sporadic Amyotrophic Lateral Sclerosis patients (SALS-CSF) on motor neuron-like cells to delineate the pathomechanism of SALS. Exposure of NSC-34 cells to SALS-CSF caused lower viability, reduction in differentiation and enhanced lactate dehydrogenase activity. Additionally, reduced choline acetyl transferase expression alongside intracellular aggregation of phosphorylated neurofilaments was prominently seen. The aggregates were immunopositive for ubiquitin. These findings are comparable to the pathological changes seen in the postmortem tissue of ALS patients. Unlimited supply of NSC-34 cells and their vulnerability to SALS-CSF render them to be a good bioassay system to identify new therapeutic agents conferring protection to motor neurons.

Prenatal auditory enrichment with species-specific calls and sitar music modulates expression of Bcl-2 and Bax to alter programmed cell death in developing chick auditory nuclei.

Postnatal auditory stimulation influences early perceptual learning. Previously we reported morphological effects of prenatal auditory stimulation by species-specific and sitar musical sounds on the chick brainstem auditory nuclei-nucleus magnocellularis and nucleus laminaris. At hatching, these two nuclei of auditory enriched embryos showed higher neuronal numbers, amongst other morphological changes. There were also increases in synaptophysin and syntaxin1 expressions in the sound enriched groups and modulation of the developmental expression of transcription factors c-Fos and c-Jun. We hypothesized that prenatal auditory enrichment may have reduced embryonic apoptosis in these nuclei with possible alteration of molecular mechanisms enhancing the postsynaptic neuron's ability to survive. In the present study, therefore, we examined apoptotic cell death by TUNEL technique and Bcl-2 expression using immunohistochemistry and immunoblotting. In the controls, a peak percentage in the TUNEL-positive cells was noted in the auditory nuclei at embryonic day 12, which was reduced at embryonic day 16. Bcl-2 immunoreactivity decreased from embryonic day 8 to embryonic day 12 overlapping the period of embryonic cell death in these nuclei. The stimulated groups, however, showed fewer apoptotic neurons and higher Bcl-2 level than that in the controls. On the other hand, Bax immunohistochemistry showed correlated reverse changes compared to Bcl-2 expression. Thus prenatal extra-acoustic stimulation appears to alter Bcl-2 and Bax expression to support cell survival and differentiation, thereby augmenting the development of auditory nuclei.

Developmentally regulated expression of c-Fos and c-Jun in the brainstem auditory nuclei of Gallus domesticus is modified by prenatal auditory enrichment.

Recognition of mother's voice by human neonates and behavioral responses of birds and animals to sounds experienced prenatally emphasize the role of sensory inputs in auditory system development. Spontaneous and experience driven neural activity influence the neural circuits' refinement in developing brain. However, cellular mechanisms endowing plasticity for such structural refinement during critical developmental periods are less understood. Sensory stimulation induces fluctuating expression of transcription factors (TFs) of Fos, Jun, and Krox families in the related brain nuclei to activate genes to synthesize proteins such as those needed for cytoskeletal structures, ion channels, and regeneration. To understand the cellular mechanism of response to prenatal auditory stimulation, we studied the expression of c-Fos and c-Jun in brainstem auditory nuclei, nucleus magnocellularis, and nucleus laminaris of the domestic chick. The chick brainstems, five each of E8 (embryonic day 8), E12, E16, E20, and posthatch day 1 were processed for immunohistochemistry as well as Western blotting and quantified using image analysis systems. In controls, c-Fos and c-Jun expression in both the nuclei was developmentally up-regulated. Reduced c-Fos expression and increase in c-Jun was temporarily observed between E12-16. In the stimulated groups, c-Fos expression was elevated while c-Jun showed a reduction matched to controls. This diametrically opposing pattern of c-Fos and c-Jun expression in response to stimulation is indicative of cell survival. Thus the expression of TFs in the auditory nuclei shows a relationship beyond a simple stimulation-activity-expression. While developmental signals control the expression of TFs, extra sensory stimulation modulates their expression to possibly support neuronal survival and enhance synthesis of other proteins.