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.

Effects of craniopharyngioma cyst fluid on neurons and glial cells cultured from rat brain hypothalamus.

Craniopharyngiomas (CPs) are rare, epithelial tumors of the central nervous system (CNS) that could lead to manifestation of multiple post-operative symptoms, ranging from hormonal imbalance to obesity, diabetes, visual, neurological and neurocognitive impairments. CP is more frequent in children, and has been reported in middle aged adults as well. In fact, arterial laceration and/or brain stroke which may occur following the removal of some CPs is mainly due to calcification of that CPs along with strong attachments to the blood vessels. The dense oily fluid content of CPs is reported to cause brain tissue damage, demyelination and axonal loss in the hypothalamus; however, its exact effect on different cell types of CNS is still unexplored. In this study, we have collected CP cyst fluid (CCF) from mostly young patients during surgical removal and exposed it 9-10 days in vitro to the primary cultures derived from rat brain hypothalamus for 48 h. A gradual decline in cell viability was noted with increasing concentration of CCF. Moreover, a distinct degenerative morphological transformation was observed in neurons and glial cells, including appearance of blebbing and overall reduction of the cell volume. Further, enhanced expression of Caspase-3 in neurons and glial cells exposed to CCF by immunofluorescence imaging, supported by Western blot experiment suggest CCF induced apoptosis of hypothalamic cells in culture. In this study, we have demonstrated the deleterious effects of the cyst fluid on various cell types within the tumors originating region of the brain and its surroundings for the first time. Taken together, this finding could be beneficial towards identifying the region specific toxic effects of the cyst fluid and its underlying mechanism.

Thyroid Hormone and Astrocyte Differentiation.

Thyroid hormones (THs) have important contributions to the development of the mammalian brain, targeting its actions on both neurons and glial cells. Astrocytes, which constitute about half of the glial cells, characteristically undergo dramatic changes in their morphology during development and such changes become necessary for the proper development of the brain. Interestingly, a large number of studies have suggested that THs play a profound role in such morphological maturation of the astrocytes. This review discusses the present knowledge on the mechanisms by which THs elicit progressive differentiation and maturation of the astrocytes. As a prelude, information on astrocyte morphology during development and its regulations, the role of THs in the various functions of astrocyte shall be dealt with for a thorough understanding of the subject of this review.

The transcription factor Pax6 contributes to the induction of GLT-1 expression in astrocytes through an interaction with a distal enhancer element.

The Na(+) -dependent glutamate transporter GLT-1 (EAAT2) shows selective expression in astrocytes, and neurons induce the expression of GLT-1 in astrocytes. In an unpublished analysis of GLT-1 promoter reporter mice, we identified an evolutionarily conserved domain of 467 nucleotides ~ 8 kb upstream of the GLT-1 translation start site that is required for astrocytic expression. Using in silico approaches, we identified Pax6 as a transcription factor that could contribute to the control of GLT-1 expression by binding within this region. We demonstrated the expression of Pax6 protein in astrocytes in vivo. Lentiviral transduction of astrocytes with exogenous Pax6 increased the expression of enhanced green fluorescent protein (eGFP) in astrocytes prepared from transgenic mice that use a bacterial artificial chromosome containing a large genomic region surrounding the GLT-1 gene to control expression of eGFP. It also increased GLT-1 protein and GLT-1-mediated uptake, whereas there was no effect on the levels of the other astroglial glutamate transporter, glutamate aspartate transporter (GLAST). Transduction of astrocytes with an shRNA directed against Pax6 reduced neuron-dependent induction of GLT-1 or eGFP. Finally, we confirmed Pax6 interaction with the predicted DNA-binding site in electrophoretic mobility assays and chromatin immunoprecipitation (ChIP). Together, these studies show that Pax6 contributes to the regulation of GLT-1 through an interaction with these distal elements and identify a novel role of Pax6 in astrocyte biology. The astroglial glutamate transporter GLT-1 shows selective expression in astrocytes and its expression can be induced by neurons. In this study, we demonstrate that Pax6 is expressed in astrocytes and binds to the GLT-1 promoter in vitro and in vivo. Exogenous expression of Pax6 increases GLT-1 and enhanced green fluorescent protein (eGFP) expression in astrocytes from a transgenic mouse line that uses the GLT-1 gene to drive eGFP expression, and an shRNA directed against Pax6 attenuates neuron-dependent induction of GLT-1/eGFP. We therefore conclude that Pax6 contributes to the neuron-dependent induction of GLT-1.

Thyroid Hormone-Induced Differentiation of Astrocytes is Associated with Transcriptional Upregulation of ß-arrestin-1 and ß-adrenergic Receptor-Mediated Endosomal Signaling.

Thyroid hormones (TH) promote differentiation of astrocytes. We have previously reported that a downstream role ß-adrenergic receptor (ß-AR) system in such effects of TH. Although evidences indicate strong interaction between TH and the ß-ARs, the underlying mechanism is poorly understood. In the present study, we further explored the influence of TH on ß-AR signaling during the differentiation process. Unlike ß1-AR, binding of (125)I-pindolol to ß2-AR in cell membranes was significantly decreased at 2 h of exposure to TH which came back to control values after 24 h. The initial decrease in ß2-AR in membranes resulted in a concomitant increase in ß2-AR levels in the cytosol, suggesting that TH may induce endocytosis of the receptor. qRT-PCR as well as Western blot analysis demonstrated that unlike ß-adrenergic receptor kinase (ß-ARK)1 and ß-ARK2, the messenger RNA (mRNA) and protein levels of ß-arrestin-1 in the astrocyte cultures increased on exposure to TH. Knockdown of ß-arrestin gene suggested requirement of both ß-arrestin-1 and ß-arrestin-2 isoforms during endocytosis of ß2-AR, thereby facilitating cell differentiation. Endocytic inhibitors blocked the delayed but sustained activation of p-extracellular signal-regulated kinase (ERK) observed during cell differentiation. Observations suggest that TH upregulate ß-arrestin-1 in astrocytes to facilitate endocytosis of ß2-AR, required for endosomal ERK activation to drive the differentiation process.

Nuclear factor-κB contributes to neuron-dependent induction of glutamate transporter-1 expression in astrocytes.

The glutamate transporter-1 [GLT-1 (excitatory amino acid transporter 2)] subtype of glutamate transporter ensures crisp excitatory signaling and limits excitotoxicity in the CNS. Astrocytic expression of GLT-1 is regulated during development, by neuronal activity, and in neurodegenerative diseases. Although neurons activate astrocytic expression of GLT-1, the mechanisms involved have not been identified. In the present study, astrocytes from transgenic mice that express enhanced green fluorescent protein (eGFP) under the control of a bacterial artificial chromosome (BAC) containing a very large region of DNA surrounding the GLT-1 gene (BAC GLT-1 eGFP mice) were used to assess the role of nuclear factor-κB (NF-κB) in neuron-dependent activation of the GLT-1 promoter. We provide evidence that neurons activate NF-κB signaling in astrocytes. Transduction of astrocytes from the BAC GLT-1 eGFP mice with dominant-negative inhibitors of NF-κB signaling completely blocked neuron-dependent activation of a NF-κB reporter construct and attenuated induction of eGFP. Exogenous expression of p65 and/or p50 NF-κB subunits induced expression of eGFP or GLT-1 and increased GLT-1-mediated transport activity. Using wild-type and mutant GLT-1 promoter reporter constructs, we found that NF-κB sites at -583 or -251 relative to the transcription start site were required for neuron-dependent reporter activation. Electrophoretic mobility shift and supershift assays reveal that p65 and p50 interact with these same sites ex vivo. Finally, chromatin immunoprecipitation showed that p65 and p50 interact with these sites in adult cortex, but not in kidney (a tissue that expresses no detectable GLT-1). Together, these studies strongly suggest that NF-κB contributes to neuron-dependent regulation of astrocytic GLT-1 transcription.

Increased beta(2)-adrenergic receptor activity by thyroid hormone possibly leads to differentiation and maturation of astrocytes in culture.

(1) Our earlier studies indicate a downsteam regulatory role of the beta-adrenergic receptor (beta-AR) system in thyroid hormone induced differentiation and maturation of astrocytes. In the present study we have investigated the contributions of the subtypes of beta-AR in the above phenomenon. (2) Primary astrocyte cultures were grown under thyroid hormone deficient as well as under euthyroid conditions. [(125)I]Pindolol ([(125)I]PIN) binding studies showed a gradual increase in the specific binding to beta(2)-AR when observed at 5, 10, 15, and 20 days under both cultural conditions. Thyroid hormone caused an increase in binding of [(125)I]PIN to beta(2)-AR compared to thyroid hormone deficient controls at all ages of astrocyte culture. (3) Saturation studies using [(125)I]PIN in astrocyte membranes prepared from 20-day-old cultures showed a significant increase in the affinity of the receptors (K (D)) in the thyroid hormone treated cells without any change in receptor number (B (max)). (4) beta(2)-AR mRNA levels were measured by real-time PCR during ontogenic development as well as during exposure of 10-day-old hypothyroid cultures to normal levels of thyroid hormone for 2, 6, 12, and 24 h. None of the conditions caused any significant change in the beta(2)-adrenergic receptor mRNA levels when compared with corresponding hypothyroid controls. (5) Over expression of beta(2)-AR cDNA in hypothyroid astrocytes caused morphological transformation in spite of the absence of thyroid hormone in the medium. (6) Taken together, results suggest thyroid hormone causes a selective increase in [(125)I]PIN binding to beta(2)-AR due to increase in receptor affinity, which may lead to maturation of astrocytes.

Thyroid hormone-induced morphological differentiation and maturation of astrocytes involves activation of protein kinase A and ERK signalling pathway.

Thyroid hormone (TH) has a profound effect on astrocyte differentiation and maturation. Astrocytes cultured under TH-deficient conditions fail to transform from flat polygonal morphology to mature, process-bearing, stellate cells. Supplementation of physiological concentrations of TH initiate gradual transformation of the cells and the process takes approximately 48 h to complete. The signal transduction pathways associated with TH-mediated maturation of astrocytes have been investigated. TH treatment caused an initial activation of protein kinase A (PKA), with a peak activity at 2 h which fell back to basal level there after. Although there was no visible change in morphology of the cells during the observed activation of PKA, it was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. PKA inhibitors as well as the MEK inhibitor PD098059 attenuated the TH-induced morphological transformation. Further studies showed that TH treatment resulted in a biphasic response on the cellular phospho-MAP kinase (p-MAPK or p-ERK) level: an initial decline in the p-ERK level followed by an induction at 18-24 h, both of which could be blocked by a PKA inhibitor. Such sustained activation of p-ERK levels by TH at this later stage coincided with initiation of morphological differentiation of the astrocytes and appeared to be critical for the transformation of astrocytes. The nitric oxide synthase (NOS) inhibitor 7-NI inhibited this induction of p-ERK activity. Moreover, the induction was accompanied by a parallel increase in phospho-CREB activity which, however, persisted at the end of the transformation of the astroglial cells.