Involvement of cystatin C in pathophysiology of CNS diseases.

Cystatin C Leu68Gln variant is known to induce amyloid deposition in cerebral arterioles, resulting in Icelandic type cerebral amyloid angiopathy (CAA). Wild-type cystatin C is also observed in solitary CAA involving amyloid beta protein (Abeta), and accelerates the amyloidogenicity of Abeta in vitro. In neurological inflammatory diseases and leptomeningeal metastasis, low cystatin C levels are accompanied with high activities of cathepsins in the cerebrospinal fluid. Among the cells in CNS, astrocytes appear to secrete cystatin C in response to various proteases and cytokines. Co-localization of Abeta and cystatin C in the brains of Alzheimer's disease (AD) led to the hypothesis that cystatin C is involved in the disease process. We demonstrated that cystatin C microinjection into rat hippocampus induced neuronal cell death in dentate gyrus. Furthermore, apoptotic cell death was observed in neuronal cells treated with cystatin C in vitro. Up-regulation of cystatin C was observed in glial cells with neuronal cell death in vivo. These findings indicate the involvement of cystatin C in the process of neuronal cell death.

A gel-based proteomic comparison of human cerebrospinal fluid between inflicted and non-inflicted pediatric traumatic brain injury.

Traumatic brain injury (TBI) is the most common cause of traumatic death in infancy, and inflicted TBI (iTBI) is the predominant cause. Like other central nervous system pathologies, TBI changes the composition of cerebrospinal fluid (CSF), which may represent a unique clinical window on brain pathophysiology. Proteomic analysis, including two-dimensional (2-D) difference in gel electrophoresis (DIGE) combined with mass spectrometry (MS), was used to compare the CSF protein profile of two pooled samples from pediatric iTBI (n = 13) and non-inflicted TBI (nTBI; n = 13) patients with severe injury. CSF proteins from iTBI and nTBI were fluorescently labeled in triplicate using different fluorescent Cy dyes and separated by 2-D gel electrophoresis. Approximately 250 protein spots were found in CSF, with 90% between-gel reproducibility of the 2-D gel. Following in-gel digestion, the tryptic peptides were analyzed by MS for protein identification. The acute phase reactant, haptoglobin (HP) isoforms, showed an approximate fourfold increase in nTBI versus iTBI. In contrast, the levels of prostaglandin D(2) synthase (PGDS) and cystatin C (CC) were 12-fold and sevenfold higher in iTBI versus nTBI, respectively. The changes of HP, PGDS, and CC were confirmed by Western blot. These initial results with conventional gel-based proteomics show new protein changes that may ultimately help to understand pathophysiological differences between iTBI and nTBI.

A novel panel of cerebrospinal fluid biomarkers for the differential diagnosis of Alzheimer's disease versus normal aging and frontotemporal dementia.

BACKGROUND: An early and accurate diagnosis of Alzheimer's disease (AD) is important in order to initiate symptomatic treatment with currently approved drugs and will be of even greater importance with the advent of disease-modifying compounds. METHODS: Protein profiles of human cerebrospinal fluid samples from patients with AD (n = 85), frontotemporal dementia (n = 20), and healthy controls (n = 32) were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to verify previously discovered biomarkers. RESULTS: We verified 15 protein biomarkers that were able to differentiate between AD and controls, and 7 of these 15 markers also differentiated AD from FTD. CONCLUSION: A panel of cerebrospinal fluid protein markers was verified by a proteomics technology which may potentially improve the accuracy of the AD diagnosis.

Cystatin F is a biomarker of prion pathogenesis in mice.

Misfolding of the cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) results in progressive, fatal, transmissible neurodegenerative conditions termed prion diseases. Experimental and epidemiological evidence point toward a protracted, clinically silent phase in prion diseases, yet there is no diagnostic test capable of identifying asymptomatic individuals incubating prions. In an effort to identify early biomarkers of prion diseases, we have compared global transcriptional profiles in brains from pre-symptomatic prion-infected mice and controls. We identified Cst7, which encodes cystatin F, as the most strongly upregulated transcript in this model. Early and robust upregulation of Cst7 mRNA levels and of its cognate protein was validated in additional mouse models of prion disease. Surprisingly, we found no significant increase in cystatin F levels in both cerebrospinal fluid or brain parenchyma of patients with Creutzfeldt-Jakob disease compared to Alzheimer's disease or non-demented controls. Our results validate cystatin F as a useful biomarker of early pathogenesis in experimental models of prion disease, and point to unexpected species-specific differences in the transcriptional responses to prion infections.

Searching for markers of Creutzfeldt-Jakob disease in cerebrospinal fluid by two-dimensional mapping.

Differential proteomic analysis has been performed on the cerebrospinal fluid (CSF) of six healthy and six patients suffering form sporadic Creutzfeldt-Jakob disease (sCJD), age- and sex-matched, after immuno-subtraction of albumin and immunoglobulins. These maps have revealed 28 polypeptide chains differentially modulated in the sCJD samples, of which 10 appeared to be up-regulated, the remaining 18 being down-regulated. Among those, 13 could be identified upon digestion and MALDI-TOF, MS analysis. In addition, the strong modulation of cystatin C was also confirmed by immunoblot analysis and the highly altered level of the 14-3-3 proteins that escaped detection by 2-D mapping, could be assessed by Western blots and immuno-detection of monomeric and homo- and hetero-dimeric 14-3-3 isotypes. In search for a panel of potential markers for sCJD, we highlight cystatin C, 14-3-3 proteins, transferrin, ubiquitin, Apoliprotein J and perhaps some of the still unidentified, but strongly modulated polypeptide chains detected in the differential map.

Molecular cloning and N-terminal analysis of bovine cystatin C. Identification of a full-length N-terminal region.

The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.

Applications of new laboratory marker assays in neurological diagnoses - a pilot study.

225 consecutive patients with different neurological diseases and 101 individuals as the control were examined between 2002-2004. Cystatin C, arginase-I, tau-protein and beta-amyloid were measured. Individuals with CNS inflammation had significantly lower Cystatin-C index (CSF/serum) values. There was no diagnostic significance of the Arginase-I assay in CSF was verified. The CSF tau-protein/beta-amyloid index was shown to be a sufficient efficacy for neurodegenerative disease diagnosis.

Alteration of cystatin C in cerebrospinal fluid of patients with sciatica revealed by a proteomical approach.

OBJECTIVE: To better understand the pathophysiological mechanisms underlying sciatica induced by lumbar intervertebral disk herniation and to ascertain the protein that presents with the most observable changes in the cerebrospinal fluid (CSF) of patients with sciatica. METHODS: We conducted the study in the Key Laboratory of Shanghai 6th People's Hospital, Shanghai Jiaotong University, Shanghai, Peoples Republic of China, during the period June 2004 to March 2005. In 2 separate experiments, we carried out the study involving the CSF of sciatica patients (the case group) and the CSF of otherwise, healthy volunteers (the control group). We utilized a proteomical analysis to compare the samples of 10 patients with sciatica with 10 volunteers in the control group. We individually separated each of the groups' CSF by 2-dimensional gel electrophoresis. We analyzed the harvested gel images with PD Quest 2D-gel software (Bio-Rad) to ascertain the differential proteins between the 2 groups. We based the enzyme linked immuno- absorbent assay (ELISA) experiment, which followed, on the results of the first experiment. RESULTS: We found 15 of the protein spots in the CSF differed appreciably in varying degrees between the 2 groups, and identification made by LC-MS/MS revealed that the most significant disparity was with cystatin C. The result of ELISA experiment confirmed a considerable decrease in the level of cystatin C (p<0.01) in the patients with sciatica. CONCLUSION: In the CSF of patients with sciatica, the volume of cystatin C increased markedly indicating that it may play an important role in the pathophysiological processes of sciatica.

Cystatin C and cathepsin B in CSF from patients with inflammatory neurologic diseases.

BACKGROUND: In CSF, proteolytic enzymes are believed to have crucial roles in the initiation and progression of inflammatory neurologic diseases (IND). Cystatin C, a major cysteine protease inhibitor in CSF, is tightly bound to cathepsin B and H. OBJECTIVE: To determine if cystatin C is involved in the disease process of IND, the authors measured the cystatin C concentration by ELISA method and cathepsin B and H activities in the CSF of patients with acute IND. METHODS: Cystatin C concentration and cathepsin B and H activities were measured in CSF samples taken from patients during the acute phase of their disease. Subjects studied were 8 patients with Guillain-Barré syndrome (GBS), 5 with chronic inflammatory demyelinating polyneuropathy (CIDP), 12 with MS, 16 with aseptic meningitis, 15 with neurodegenerative diseases as disease controls, and 35 healthy controls. RESULTS: A significant decrease in CSF cystatin C level was seen in the patients with GBS, CIDP, and MS compared to the control subjects. High cathepsin B activity, but not cathepsin H activity, was also observed in the patients with GBS, CIDP, and MS. CONCLUSION: Cystatin C levels in CSF measured by ELISA may help the physician recognize GBS, CIDP, and MS. Decreased levels of cystatin C may be related to the high levels of cathepsin B activity seen in the CSF of patients with GBS, CIDP, and MS.

Cystatin C as a cerebrospinal fluid biomarker for pain in humans.

Through a process of subtraction cloning and differential hybridization, we previously identified several new genes whose expression was induced by peripheral inflammation. One of these coded for cystatin C, a secreted cysteine protease inhibitor in the cystatin superfamily. We hypothesized that, concurrent with increased expression in dorsal horn, increased secretion would elevate the cystatin C content in cerebrospinal fluid (CSF) during active pain states. Alterations were assessed by immunoassay and by surface enhanced laser desorption ionization (SELDI) mass spectrometry with either reverse phase or immobilized anti-cystatin C antibody surfaces using CSF from ten age-matched obstetrical patients at term. Five control subjects were scheduled for an elective caesarian section and were not in pain. Another five subjects were in labor for 8.9+/-1h and were in severe pain as assessed with a visual analog scale and the McGill short form questionnaire. The level of cystatin C as measured by immunoassay in the non-pain patients was 2.77+/-0.75 microg/ml and in the pain patients 5.36+/-0.92 microg/ml (P<0.02). The elevation occurred without significant change in total CSF protein or beta-endorphin content. The cystatin C increase also was detectable by SELDI with either raw CSF or after antibody capture. These data are consistent with our previous animal study and the idea that persistent pain induces the synthesis and release of cystatin C in dorsal spinal cord, the surplus of which overflows into the CSF.

Cystatin C in cerebrospinal fluid is not a diagnostic test for pain in humans.

A recent subtractive cDNA cloning study in rats demonstrated an unexpected increase in expression of the proteinase inhibitor, cystatin C in the spinal cord during acute peripheral inflammation, suggesting this protein may be involved in the pathogenesis of persistent pain. A subsequent study of 10 women suggested that prolonged labor pain resulted in increased cystatin C concentrations in cerebrospinal fluid, and that this could be used as a biomarker for pain. To confirm and extend these observations, we measured cystatin C concentrations in cerebrospinal fluid in 131 subjects: 30 normal volunteers without pain, 25 women at elective cesarean section without pain, 60 women in labor with severe pain, and 16 patients with chronic neuropathic pain and tactile allodynia. The median cystatin C concentration in normal volunteers, 2.2 microg/ml, was similar to that previously reported by multiple investigators, and cystatin C concentrations were increased in women in labor (3.9 microg/ml). However, contrary to the previous report, cystatin C concentrations in laboring women with pain did not differ from those of pregnant women without pain (3.7 microg/ml). There was no relationship between duration of painful labor and cystatin C concentration. Patients with neuropathic pain had similar cystatin C concentrations (2.4 microg/ml) to controls. Logistic regression analysis indicated that cystatin C concentrations could not be used to reliably predict the presence of pain in either acute or chronic settings. These data suggest that cystatin C concentration in cerebrospinal fluid is an unreliable diagnostic marker for pain in humans.

Localization of cystatin mRNA in chicken brain by in situ hybridization.

The cystatins are a superfamily of proteins that inhibit the lysosomal cysteine proteinases, cathepsins B, H, and L. Members of this superfamily have been found in all tissues and biological fluids analyzed. Previous studies have shown that chicken cystatin mRNA is abundant in brain tissue. In this study, a definitive localization of chicken cystatin mRNA in chicken brain was determined by in situ hybridization. Chicken cystatin mRNA was heavily concentrated in the secretory epithelial cells of the choroid plexus. The rest of the brain failed to show a hybridization signal above that of the control sense strand probe, even after long exposures. We conclude that chicken cystatin is synthesized predominantly by the specialized secretory epithelial cells of the choroid plexus and secreted into the cerebrospinal fluid (CSF). We postulate that chicken cystatin functions to regulate proteinase activity in the CSF and therefore may function as a protective factor for the cellular elements of the central nervous system.

The high alkaline fraction on isoelectric focusing of cerebrospinal fluid is cystatin C.

A high alkaline fraction with a pI of 9.2 is sometimes seen on isoelectric focusing patterns of cerebrospinal fluid. The appearance of this fraction mainly depends on the type of concentrators used to prepare the cerebrospinal fluid samples, prior to isoelectric focusing. The amino acid sequence of the high alkaline fraction showed sequence identity to cystatin C, a cysteine protease inhibitor with a pI of 9.2-9.3 and a molecular mass of 13.4 kDa. In addition, on Western blot the high alkaline fraction was recognized by an antibody, directed against cystatin C. Taken together, the present findings demonstrate that the high alkaline fraction is cystatin C.

Dynamics of brain-derived proteins in cerebrospinal fluid.

BACKGROUND: The recent theory of blood-cerebrospinal fluid (CSF) barrier function and dysfunction connects molecular flux and CSF flow rate. A reduced CSF flow rate is sufficient to account for the observed hyperbolic relation between different blood-derived protein concentrations in CSF in cases of a blood-CSF barrier dysfunction. METHODS: The dynamics of brain-derived proteins in CSF are investigated with reference to the CSF flow rate measured by CSF/serum albumin concentration quotient. RESULTS: Proteins from neurons or glial cells, tau protein, neuron-specific enolase, S-100 protein, all enter CSF primarily in the ventricular and cisternal space. Their concentration between normal ventricular and lumbar CSF is decreasing (in contrast to blood-derived proteins), and in the case of pathologically decreasing CSF flow rate, the concentration in lumbar CSF remains invariantly constant. Concentrations of the primarily leptomeningeal proteins, beta-trace protein and cystatin C, increase between normal ventricular and lumbar CSF, and in the case of pathologically decreased CSF flow rate they increase linearly in lumbar CSF (concentrations of blood-derived proteins increase non-linearly). CONCLUSIONS: A satisfactory physiological explanation can now be given for the dynamics of proteins in CSF consisting of both brain- and blood-derived fractions (transthyretin, soluble intercellular adhesion molecule (s-ICAM)), as well as the disputed decrease of leptomeningeal protein concentrations (beta-trace protein, cystatin C) in cases of bacterial meningitis is also explained. The biophysical treatment of dynamics in the ventricular and lumbar CSF extends the new theory and shows that CSF flow rate is the most relevant parameter for understanding the pathological changes of both blood- and brain-derived proteins in CSF. The impact on diagnosis of neuro-degenerative diseases is discussed.

Evaluation of cardiac troponin I and T levels as markers of myocardial damage in doxorubicin-induced cardiomyopathy rats, and their relationship with echocardiographic and histological findings.

BACKGROUND: Cardiac troponins I (cTnI) and T (cTnT) have been shown to be highly sensitive and specific markers of myocardial cell injury. We investigated the diagnostic value of cTnI and cTnT for the diagnosis of myocardial damage in a rat model of doxorubicin (DOX)-induced cardiomyopathy, and we examined the relationship between serial cTnI and cTnT with the development of cardiac disorders monitored by echocardiography and histological examinations in this model. METHODS: Thirty-five Wistar rats were given 1.5 mg/kg DOX, i.v., weekly for up to 8 weeks for a total cumulative dose of 12 mg/kg BW. Ten rats received saline as a control group. cTnI was measured with Access(R) (ng/ml) and a research immunoassay (pg/ml), and compared with cTnT, CK-MB mass and CK. By using transthoracic echocardiography, anterior and posterior wall thickness, LV diameters and LV fractional shortening (FS) were measured in all rats before DOX or saline, and at weeks 6 and 9 after treatment in all surviving rats. Histology was performed in DOX-rats at 6 and 9 weeks after the last DOX dose and in all controls. RESULTS: Eighteen of the DOX rats died prematurely of general toxicity during the 9-week period. End-diastolic (ED) and end-systolic (ES) LV diameters/BW significantly increased, whereas LV FS was decreased after 9 weeks in the DOX group (p<0.001). These parameters remained unchanged in controls. Histological evaluation of hearts from all rats given DOX revealed significant slight degrees of perivascular and interstitial fibrosis. In 7 of the 18 rats, degeneration and myocyte vacuolisation were found. Only five of the controls exhibited evidence of very slight perivascular fibrosis. A significant rise in cTnT was found in DOX rats after cumulative doses of 7.5 and 12 mg/kg in comparison with baseline (p<0.05). cTnT found in rats after 12 mg/kg were significantly greater than that found after 7.5 mg/kg DOX. Maximal cTnI (pg/ml) and cTnT levels were significantly increased in DOX rats compared with controls (p=0.006, 0.007). cTnI (ng/ml), CK-MB mass and CK remained unchanged in DOX rats compared with controls. All markers remained stable in controls. Analysis of data revealed a significant correlation between maximal cTnT and ED and ES LV diameters/BW (r=0.81 and 0.65; p<0.0001). A significant relationship was observed between maximal cTnT and the extent of myocardial morphological changes, and between LV diameters/BW and histological findings. CONCLUSIONS: Among markers of ischemic injury after DOX in rats, cTnT showed the greatest ability to detect myocardial damage assessed by echocardiographic detection and histological changes. Although there was a discrepancy between the amount of cTnI and cTnT after DOX, probably due to heterogeneity in cross-reactivities of mAbs to various cTnI and cTnT forms, it is likely that cTnT in rats after DOX indicates cell damage determined by the magnitude of injury induced and that cTnT should be a useful marker for the prediction of experimentally induced cardiotoxicity and possibly for cardioprotective experiments.

Cathepsin B and H activities and cystatin C concentrations in cerebrospinal fluid from patients with leptomeningeal metastasis.

BACKGROUND: Cysteine proteases are involved in the extension of cancer into the subarachnoid space. The presence of cathepsins B and H along with their potent inhibitor cystatin C in the cerebrospinal fluid (CSF) was investigated in patients with leptomeningeal metastasis of cancer (LM). MATERIALS AND METHODS: CSF samples were obtained in 16 cases of LM (10 solid tumors and 6 leukemia or lymphoma) and compared with 11 cancer cases without involvement of the central nervous system, 12 multiple sclerosis cases and 34 healthy volunteers. The activity of the enzymes was measured, their molecular forms were analyzed by the Western blotting, and the concentration of cystatin C was measured by ELISA. Immunohistochemistry of the leptomeningeal tissues was also performed in six autopsy cases of LM. RESULTS: High activities of cathepsins B and H along with decreased cystatin C concentration were observed in CSF of LM as compared with three control groups. Western blot analysis revealed higher concentration of the enzyme protein as well as its active forms in samples with higher enzyme activity. Cells metastasizing leptomeningeal tissue were clearly positive in immunohistochemical staining of cathepsins, indicating active production by tumor cells. CONCLUSION: Production of cathepsins B and H by tumor cells and their high activity along with concomitant decrease of their potent inhibitor, cystatin C, in the CSF might contribute in the process of metastasis and spread of the cancer cells in the leptomeningeal tissues. A high enzyme activity/cystatin C concentration ratio in the CSF could be useful when diagnosing LM in combination with other parameters.

Simultaneous multiple brain hemorrhage associated with migraine--a case report.

A fifty-five-year-old woman with a history of migraine suddenly developed an occipital headache and visual disturbance after a typical migrainous attack. On admission, she had a left homonymous hemianopsia, and computed tomography of the brain demonstrated intracranial hematomas in the occipital subcortices bilaterally. Cerebral arteriography revealed diffuse vasospasm of the intracranial arteries attributed to the migraine. The cystatin C concentration in the cerebrospinal fluid was low, which suggested the existence of cerebral amyloid angiopathy. According to the clinical course and angiographic findings, it is suggested that the vasospasm associated with migraine played an important role in developing multiple brain hemorrhage in this patient.

[A case of lobar cerebral hemorrhage with low concentration of CSF cystatin C].

A 49-year-old male was admitted to our hospital because of severe headache and dizziness which had occurred suddenly one day before admission. There was no past history contributory to cerebral hemorrhage but was family history of cerebrovascular accidents in his father and brother. Neurological examination revealed left homonymous hemianopsia, mild left hemiparesis, and left side hemi-neglect in simultaneous stimuli on bilateral extremities. Laboratory data including peripheral blood cells, coagulation tests, and serum chemistry were unremarkable. Brain CT and MRI demonstrated large lobar hematoma in the right parieto-occipito-temporal region. Cystatin C level in the CSF samples taken on the 39th and 59 th days (38 and 27 ng/ml respectively) were low, compared with the normal value (greater than 100 ng/ml). These findings suggest that the lobar cerebral hemorrhage of the present case might have been caused by cerebral amyloid angiopathy with cystatin C deposits.

[Diagnosis of cerebral amyloid angiopathy with cerebral hemorrhage by enzyme linked immunosorbent assay (ELISA) of cystatin C in the cerebrospinal fluid].

The lower level of cystatin C in cerebrospinal fluid (CSF) is one of the useful diagnostic markers of hereditary cerebral hemorrhage with amyloidosis in Iceland. We attempted to establish an assay to determine the level of cystatin C in CSF for diagnosis of CAA due to the deposition of cystatin C in CSF for diagnosis of CAA due to the deposition of cystatin C. We carried out the sandwich enzyme immunosorbent assay with the use of monoclonal mouse anti-cystatin C and polyclonal rabbit anti-cystatin C antibodies. CSF from nine cases of cerebral hemorrhage and fifty reference cases with other neurological diseases were examined. Four patients with cerebral hemorrhage showed a low level of cystatin C and clinical manifestations suggestive of CAA. Our study showed the feasibility of using ELISA for the diagnosis of cerebral amyloid angiopathy that causes cerebral hemorrhage with the deposition of cystatin C.

Measurement of cystatin C in cerebrospinal fluid.

Cyctatin C(CysC), a cysteine proteinase inhibitor, is produced from most cells and exists in various body fluids. We measured CysC concentrations in cerebrospinal fluid(CSF) using the same assay method as for serum CysC measurement. CSF-CysC concentrations in subjects without apparent disorders ranged from 1.8 to 7.2 mg/l(2SD) with a mean of 3.6 mg/l. Because serum CysC concentrations are less than 1 mg/l, CSF-CysC may preferably represent those produced in central nervous system. CysC was measured in CSF samples obtained in a hospital laboratory and their clinical information was retrospectively viewed. Generally, none of various disease groups, including infection, neoplasma, and vascular diseases had CysC values significantly different from the normal reference values. Subjects undergoing surgery and with hydrocephalus had varied CysC values, raising the possibility that CysC values may change under the influence of CSF flow.