Therapeutic Options of Crystallin Mu and Protein Disulfide Isomerase A3 for Cuprizone-Induced Demyelination in Mouse Hippocampus.

This study investigates the changes in hippocampal proteomic profiles during demyelination and remyelination using the cuprizone model. Employing two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry for protein profiling, we observed significant alterations in the expression of ketimine reductase mu-crystallin (CRYM) and protein disulfide isomerase A3 precursor (PDIA3) following exposure to and subsequent withdrawal from cuprizone. Immunohistochemical staining validated these protein expression patterns in the hippocampus, revealing that both PDIA3 and CRYM were downregulated in the hippocampal CA1 region during demyelination and upregulated during remyelination. Additionally, we explored the potential protective effects of CRYM and PDIA3 against cuprizone-induced demyelination by synthesizing cell-permeable Tat peptide-fusion proteins (Tat-CRYM and Tat-PDIA3) to facilitate their crossing through the blood-brain barrier. Our results indicated that administering Tat-CRYM and Tat-PDIA3 mitigated the reduction in proliferating cell and differentiated neuroblast counts compared to the group receiving cuprizone alone. Notably, Tat-PDIA3 demonstrated significant effects in enhancing myelin basic protein expression alongside phosphorylation of CREB in the hippocampus, suggesting its potential therapeutic role in the prevention or treatment of demyelination, and by extension, in conditions such as multiple sclerosis.

Gene expression patterns of CRYM and SIGLEC10 in Alzheimer's disease: potential early diagnostic indicators.

BACKGROUND: Alzheimer's disease (AD) is a neurological condition that may lead to dementia as well as a slow and steady decline in cognitive ability. Finding early signs that may be used in the diagnosis of AD is still a difficult aim to achieve in the field of medical practice. METHODS AND RESULTS: The purpose of this research was to investigate to determine any differences in the gene expression patterns of crystallin mu (CRYM) and sialic acid-binding immunoglobulin-like lectin 10 (SIGLEC10) in whole blood samples obtained from fifty individuals who were diagnosed with AD and fifty individuals as a control group. When compared with controls, it was discovered that the expression of the CRYM gene was substantially decreased in AD patients, but the expression of the SIGLEC10 gene was significantly higher. A positive correlation between CRYM and SIGLEC10 was noticed solely in patients with AD. Furthermore, assessing the diagnostic value of these genes, CRYM and SIGLEC10 transcript levels displayed an area under the curve (AUC) of 0.74 and 0.81, respectively. CONCLUSIONS: These results suggest that alterations in CRYM and SIGLEC10 expression may be implicated in AD pathology and that these genes expression levels can potentially serve as biomarkers for early detection and diagnosis of AD. Nevertheless, further validation of these findings requires the inclusion of more extensive and heterogeneous cohorts. The findings derived from this study possess the capability to offer a significant contribution towards the progression of innovative diagnostic and therapeutic strategies for AD.

µ-Crystallin: A thyroid hormone binding protein.

µ-Crystallin is a NADPH-regulated thyroid hormone binding protein encoded by the CRYM gene in humans. It is primarily expressed in the brain, muscle, prostate, and kidney, where it binds thyroid hormones, which regulate metabolism and thermogenesis. It also acts as a ketimine reductase in the lysine degradation pathway when it is not bound to thyroid hormone. Mutations in CRYM can result in non-syndromic deafness, while its aberrant expression, predominantly in the brain but also in other tissues, has been associated with psychiatric, neuromuscular, and inflammatory diseases. CRYM expression is highly variable in human skeletal muscle, with 15% of individuals expressing ≥13 fold more CRYM mRNA than the median level. Ablation of the Crym gene in murine models results in the hypertrophy of fast twitch muscle fibers and an increase in fat mass of mice fed a high fat diet. Overexpression of Crym in mice causes a shift in energy utilization away from glycolysis towards an increase in the catabolism of fat via ß-oxidation, with commensurate changes of metabolically involved transcripts and proteins. The history, attributes, functions, and diseases associated with CRYM, an important modulator of metabolism, are reviewed.

Loss of µ-crystallin causes PPARγ activation and obesity in high-fat diet-fed mice.

The thyroid hormone-binding protein µ-crystallin (CRYM) mediates thyroid hormone action by sequestering triiodothyronine in the cytoplasm and regulating the intracellular concentration of thyroid hormone. As thyroid hormone action is closely associated with glycolipid metabolism, it has been proposed that CRYM may contribute to this process by reserving or releasing triiodothyronine in the cytoplasm. We aimed to clarify the relationship between CRYM and glycolipid metabolism by comparing wild-type and CRYM knockout mice fed a high-fat diet. Each group was provided a high-fat diet for 10 weeks, and then their body weight and fasting blood glucose levels were measured. Although no difference in body weight was observed between the two groups with normal diet, the treatment with a high-fat diet was found to induce obesity in the knockout mice. The knockout group displayed increased dietary intake, white adipose tissue, fat cell hypertrophy, and hyperglycemia in the intraperitoneal glucose tolerance test. In CRYM knockout mice, liver fat deposits were more pronounced than in the control group. Enhanced levels of PPARγ, which is known to cause fatty liver, and ACC1, which is a target gene for thyroid hormone and is involved in the fat synthesis, were also detected in the livers of CRYM knockout mice. These observations suggest that CRYM deficiency leads to obesity and lipogenesis, possibly in part through increasing the food intake of mice fed a high-fat diet.

Ketimine reductase/CRYM catalyzes reductive alkylamination of α-keto acids, confirming its function as an imine reductase.

Recently, crystalized mouse ketimine reductase/CRYM complexed with NADPH was found to have pyruvate bound in its active site. We demonstrate that the enzyme binds α-keto acids, such as pyruvate, in solution, and catalyzes the formation of N-alkyl-amino acids from alkylamines and α-keto acids (via reduction of imine intermediates), but at concentrations of these compounds not expected to be encountered in vivo. These findings confirm that, mechanistically, ketimine reductase/CRYM acts as a classical imine reductase and may explain the finding of bound pyruvate in the crystallized protein.

LncRNA CRYM-AS1 Inhibits Gastric Cancer Progression via Epigenetically Regulating CRYM.

OBJECTIVE: To study the role of long non-coding RNA (lncRNA) CRYM-AS1 in human gastric cancer. METHODS: Expression levels of CRYM-AS1 in cell lines and clinical tissues were examined by RT-qPCR. The association between CRYM-AS1 levels and clinicopathological parameters/survival rates of gastric cancer patients was analyzed. Cell functional experiments including MTT assay, glucose consumption/lactate production/ATP production detection was performed to examine the role of CRYM-AS1 in cell aerobic glycolysis and cell proliferation of gastric cancer cells. Subcellular fractionation location detection, western blot, RIP (RNA binding protein immunoprecipitation) assay, CHIP (Chromatin immunoprecipitation) assay, and BSP (Bisulfite sequencing PCR) assay were carried out to explore the molecular mechanism of CRYM-AS1 in gastric cancer cells. RESULTS: CRYM-AS1 was low expressed in gastric cancer cells and tissues compared with normal gastric cells and tissues respectively. CRYM-AS1 was negatively correlated with TNM staging, tumor size, and overall survival (OS) rate in gastric cancer patients. CRYM-AS1 inhibited gastric cancer cell aerobic glycolysis and cell proliferation. CRYM-AS1 directly bound to EZH2 and mediated the CRYM promoter methylation and consequently negatively regulated the expression of CRYM. Forced expression of CRYM rescued the decreased aerobic glycolysis and cell proliferation induced by CRYM-AS1 in gastric cancer cells. CONCLUSION: CRYM-AS1 was an important biomarker and could be used for human gastric cancer treatment.

Emerging role of T3-binding protein µ-crystallin (CRYM) in health and disease.

Thyroid hormones are essential metabolic and developmental regulators that exert a huge variety of effects in different organs. Triiodothyronine (T3) and thyroxine (T4) are synthesized in the thyroid gland and constitute unique iodine-containing hormones that are constantly regulated by a homeostatic feedback mechanism. T3/T4 activity in cells is mainly determined by specific transporters, cytosolic binding proteins, deiodinases (DIOs), and nuclear receptors. Modulation of intracellular T3/T4 level contributes to the maintenance of this regulatory feedback. µ-Crystallin (CRYM) is an important intracellular high-affinity T3-binding protein that buffers the amount of T3 freely available in the cytosol, thereby controlling its action. In this review, we focus on the molecular and pathological properties of CRYM in thyroid hormone signaling, with emphasis on its critical role in malignancies.

Prognostic Relevance of Thyroid-Hormone-Associated Proteins in Adenoid Cystic Carcinoma of the Head and Neck.

The proteins sodium iodide symporter (NIS), µ-crystallin (CRYM), and thyroid hormone receptor beta (THRB) have been associated with prognosis in various cancer entities. While NIS and THRB may serve as possible therapeutic targets, the role of CRYM in cancer is still unclear. Protein levels of 44 patients with adenoid cystic carcinoma of the head and neck were analyzed using immunohistochemistry and correlated with clinicopathological data and outcome. NIS was positive in 72%, CRYM was positive in 55%, and THRB was positive in 39% of the patients. CRYM-positive adenoid cystic carcinomas were associated with a better cause-specific survival. Thus, our data indicate that CRYM might be a suitable positive prognostic marker in adenoid cystic carcinoma of the head and neck. Furthermore, expression of NIS was present in most patients and therefore evaluation of the use of radioiodine treatment is recommended.

Thyroxine replacement modifies changes in deiodinase and thyroid hormone transporter expression induced by subclinical hypothyroidism in rats.

PURPOSE: The potential benefits of treating subclinical hypothyroidism (SCH) are unclear and still controversial. Thus, we surgically induced SCH in rats and evaluated the effects of thyroxine (T4) replacement on the gene expression levels of deiodinases and thyroid hormone (TH) transporters in different tissues. METHODS: SCH was induced by hemithyroid electrocauterization. The control animals underwent the same surgical procedure but were not subjected to electrocauterization (sham). After 14 days, half of the SCH animals were treated with T4 (SCH + T4). At the end of the experimental protocol, all of the rats were euthanized, serum hormone concentrations were measured, and RNA analyses were performed on different tissues and organs. RESULTS: Consistent with previous studies, we observed increased TSH levels, normal TH levels, and reduced hypothalamic TRH expression in the SCH group. Additionally, Dio2 mRNA expression was downregulated in the hippocampus and pituitary, and Dio1 was upregulated in the kidney and pituitary of the SCH animals. The changes in Dio3 expression were tissue-specific. Concerning TH transporters, Mct10 expression was upregulated in the pituitary, kidney, hypothalamus, and hippocampus, and Mct8 expression was downregulated in the kidney of the SCH group. Crym expression was upregulated in the kidney and pituitary. Notably, T4 replacement significantly attenuated serum TSH levels and reverted Dio1, Dio2, Mct10, and Crym expression in the pituitary, hippocampus, and kidney to levels that were similar to the sham group. Tissue-specific responses were also observed in the liver and hypothalamus. CONCLUSION: Our results indicate that treatment of SCH should be considered before the appearance of clinical symptoms of hypothyroidism.

Identification of a novel mutation in CRYM in a Chinese family with hearing loss using whole-exome sequencing.

Previous studies have identified ~50 genes that contribute to non-syndromic autosomal dominant sensorineural deafness (DFNA). However, in numerous families with hearing loss, the specific gene mutation remains to be identified. In the present study, the clinical characteristics and gene mutations were analyzed in a Chinese pedigree with hereditary hearing loss. The clinical characteristics of the family members were assessed and a detailed audiology function examination was performed. Whole-exome sequencing (WES) was performed to identify the gene mutation responsible for the hearing loss. Sanger sequencing was used to verify the candidate mutation detected in the family. The family consisted of 31 members, seven of whom were diagnosed with sensorineural deafness of varying degrees. No mutation was identified by the general deafness gene chip. However, a novel heterozygous mutation in exon 3 (c.152C>T; Pro51Leu) of the gene crystallin µ (CRYM) was identified by WES. This result was further verified by Sanger sequencing. Co-segregation of genotypes and phenotypes suggested that this novel mutation was instrumental for the hearing loss/DFNA. In conclusion, the present study identified a novel pathogenic mutation, NM_001888.5(CRYM): c.152C>T(Pro51Leu), associated with DFNA. This mutation has not been reported previously and further functional studies are warranted.

Gene Expression Profiling of Different Huh7 Variants Reveals Novel Hepatitis C Virus Host Factors.

Chronic Hepatitis C virus (HCV) infection still constitutes a major global health problem with almost half a million deaths per year. To date, the human hepatoma cell line Huh7 and its derivatives is the only cell line that robustly replicates HCV. However, even different subclones and passages of this single cell line exhibit tremendous differences in HCV replication efficiency. By comparative gene expression profiling using a multi-pronged correlation analysis across eight different Huh7 variants, we identified 34 candidate host factors possibly affecting HCV permissiveness. For seven of the candidates, we could show by knock-down studies their implication in HCV replication. Notably, for at least four of them, we furthermore found that overexpression boosted HCV replication in lowly permissive Huh7 cells, most prominently for the histone-binding transcriptional repressor THAP7 and the nuclear receptor NR0B2. For NR0B2, our results suggest a finely balanced expression optimum reached in highly permissive Huh7 cells, with even higher levels leading to a nearly complete breakdown of HCV replication, likely due to a dysregulation of bile acid and cholesterol metabolism. Our unbiased expression-profiling approach, hence, led to the identification of four host cellular genes that contribute to HCV permissiveness in Huh7 cells. These findings add to an improved understanding of the molecular underpinnings of the strict host cell tropism of HCV.

Ornithine cyclodeaminase/µ-crystallin homolog from the hyperthermophilic archaeon Thermococcus litoralis functions as a novel Δ(1)-pyrroline-2-carboxylate reductase involved in putative trans-3-hydroxy-l-proline metabolism.

l-Ornithine cyclodeaminase (OCD) is involved in l-proline biosynthesis and catalyzes the unique deaminating cyclization of l-ornithine to l-proline via a Δ(1)-pyrroline-2-carboxyrate (Pyr2C) intermediate. Although this pathway functions in only a few bacteria, many archaea possess OCD-like genes (proteins), among which only AF1665 protein (gene) from Archaeoglobus fulgidus has been characterized as an NAD(+)-dependent l-alanine dehydrogenase (AfAlaDH). However, the physiological role of OCD-like proteins from archaea has been unclear. Recently, we revealed that Pyr2C reductase, involved in trans-3-hydroxy-l-proline (T3LHyp) metabolism of bacteria, belongs to the OCD protein superfamily and catalyzes only the reduction of Pyr2C to l-proline (no OCD activity) [FEBS Open Bio (2014) 4, 240-250]. In this study, based on bioinformatics analysis, we assumed that the OCD-like gene from Thermococcus litoralis DSM 5473 is related to T3LHyp and/or proline metabolism (TlLhpI). Interestingly, TlLhpI showed three different enzymatic activities: AlaDH; N-methyl-l-alanine dehydrogenase; Pyr2C reductase. Kinetic analysis suggested strongly that Pyr2C is the preferred substrate. In spite of their similar activity, TlLhpI had a poor phylogenetic relationship to the bacterial and mammalian reductases for Pyr2C and formed a close but distinct subfamily to AfAlaDH, indicating convergent evolution. Introduction of several specific amino acid residues for OCD and/or AfAlaDH by site-directed mutagenesis had marked effects on both AlaDH and Pyr2C reductase activities. The OCC_00387 gene, clustered with the TlLhpI gene on the genome, encoded T3LHyp dehydratase, homologous to the bacterial and mammalian enzymes. To our knowledge, this is the first report of T3LHyp metabolism from archaea.

Multimodal exercises simultaneously stimulating cortical and brainstem pathways after unilateral corticospinal lesion.

In the context of injury to the corticospinal tract (CST), brainstem-origin circuits may provide an alternative system of descending motor influence. However, subcortical circuits are largely under subconscious control. To improve volitional control over spared fibers after CST injury, we hypothesized that a combination of physical exercises simultaneously stimulating cortical and brainstem pathways above the injury would strengthen corticobulbar connections through Hebbian-like mechanisms. We sought to test this hypothesis in mice with unilateral CST lesions. Ten days after pyramidotomy, mice were randomized to four training groups: (1) postural exercises designed to stimulate brainstem pathways (BS); (2) distal limb-grip exercises preferentially stimulating CST pathways (CST); (3) simultaneous multimodal exercises (BS+CST); or (4) no training (NT). Behavioral and anatomical outcomes were assessed after 20 training sessions over 4 weeks. Mice in the BS+CST training group showed a trend toward greater improvements in skilled limb performance than mice in the other groups. There were no consistent differences between training groups in gait kinematics. Anatomically, multimodal BS+CST training neither increased corticobulbar fiber density of the lesioned CST rostral to the lesion nor collateral sprouting of the unlesioned CST caudal to the lesion. Further studies should incorporate electrophysiological assessment to gauge changes in synaptic strength of direct and indirect pathways between the cortex and spinal cord in response to multimodal exercises.