Cerebrospinal fluid proteomics implicates the granin family in Parkinson's disease.

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Better understanding of the underlying disease mechanism(s) is an urgent need for the development of disease-modifying therapeutics. Limited studies have been performed in large patient cohorts to identify protein alterations in cerebrospinal fluid (CSF), a proximal site to pathology. We set out to identify disease-relevant protein changes in CSF to gain insights into the etiology of Parkinson's disease and potentially assist in disease biomarker identification. In this study, we used liquid chromatography-tandem mass spectrometry in data-independent acquisition (DIA) mode to identify Parkinson's-relevant biomarkers in cerebrospinal fluid. We quantified 341 protein groups in two independent cohorts (n = 196) and a longitudinal cohort (n = 105 samples, representing 40 patients) consisting of Parkinson's disease and healthy control samples from three different sources. A first cohort of 53 Parkinson's disease and 72 control samples was analyzed, identifying 53 proteins with significant changes (p < 0.05) in Parkinson's disease relative to healthy control. We established a biomarker signature and multiple protein ratios that differentiate Parkinson's disease from healthy controls and validated these results in an independent cohort. The second cohort included 28 Parkinson's disease and 43 control samples. Independent analysis of these samples identified 41 proteins with significant changes. Evaluation of the overlapping changes between the two cohorts identified 13 proteins with consistent and significant changes (p < 0.05). Importantly, we found the extended granin family proteins as reduced in disease, suggesting a potential common mechanism for the biological reduction in monoamine neurotransmission in Parkinson's patients. Our study identifies several novel protein changes in Parkinson's disease cerebrospinal fluid that may be exploited for understanding etiology of disease and for biomarker development.

CSF of neuroleptic-naive first-episode schizophrenic patients: levels of biogenic amines, substance P, and peptides derived from chromogranin A (GE-25) and secretogranin II (secretoneurin).

Lumbar cerebrospinal fluid (CSF) was collected from controls and neuroleptic-naive patients with their first acute schizophrenic episode. The CSF was analyzed for several biogenic amines and their metabolites [dopamine,dihydroxyphenylacetic acid (DOPAC), noradrenaline, 5-hydroxytryptamine (5-HT), 5-hydroxyindolacetic acid (5-HIAA)]. For these transmitters, which are stored and secreted from synaptic vesicles, there was no significant difference between controls and schizophrenic patients. As constituents of large dense-core vesicles substance P (SP) and GE-25 (derived from chromogranin A)-and secretoneurin (derived from secretogranin 11)-immunoreactivities were determined. SP-like immunoreactivity levels did not differ between controls and patients; however, GE-25 was elevated and especially the GE-25/secretoneurin ratio was significantly (p < .001) higher in patients. Characterization of the immunoreactivities by high-performance liquid chromatography did not reveal any difference between patients (n = 3) and controls in the processing of the two proproteins chromogranin A and secretogranin II. These data indicate that proteolytic processing of the two widespread constituents of large dense-core vesicles, i.e., chromogranin A and secretogranin II, is not altered in schizophrenic patients. The increase in the chromogranin A /secretoneurin ratio in schizophrenic patients deserves further investigation in order to elucidate its possible pathogenetic significance.

Cerebrospinal fluid chromogranin A is unchanged in Alzheimer dementia.

Several lines of evidence link chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, with the cholinergic nervous system, abnormalities of which may play a central role in memory deficits in Alzheimer dementia. Because of reported elevations of CgA in Alzheimer brains and its presence in the senile plaque lesions of such brains, we evaluated the concentration of CgA in cerebrospinal fluid of Alzheimer dementia patients and matched controls. CgA was detectable in each sample, but the results in dementia showed substantial overlap with and no significant (p = 0.55) difference from the results in healthy controls. We conclude that measurement of cerebrospinal fluid CgA offers no diagnostic assistance in Alzheimer dementia.

Noradrenergic activity and prediction of psychotic relapse following haloperidol withdrawal in schizophrenia.

OBJECTIVE: The purpose of this study was to develop a model based on the authors' previous studies to identify which neuroleptic-treated schizophrenic patients are at risk of early relapse following drug withdrawal. METHOD: Clinical and CSF monoamine-related variables obtained for 50 male haloperidol-treated, schizophrenic patients were used in a logistic regression model to identify those who relapsed (N = 24) within 6 weeks after placebo substitution and those who did not (N = 26). RESULTS: The oral dose of haloperidol, weight, CSF norepinephrine, 3-methoxy-4-hydroxyphenylglycol and chromogranin A-like immunoreactivity, and the anxiety and paranoia subscale ratings of the Brief Psychiatric Rating Scale produced a model that correctly predicted 18 relapsers and 21 nonrelapsers. By including the interactions of paranoia subscale by CSF norepinephrine and anxiety by CSF norepinephrine, the model correctly identified 20 relapsers and 23 nonrelapsers with a sensitivity and specificity of 83% and 88%, respectively. CONCLUSIONS: Increased noradrenergic activity during chronic dopamine blockade may be an episode marker and may predict relapse within 6 weeks following haloperidol withdrawal in schizophrenia. Effective relapse prediction models have important practical implications for the treatment of schizophrenia and the understanding of the psychotic relapse process.

Molecular characterization of immunoreactivities of peptides derived from chromogranin A (GE-25) and from secretogranin II (secretoneurin) in human and bovine cerebrospinal fluid.

Chromogranin A and secretogranin II are members of the so-called chromogranins, the acidic proteins stored in neuroendocrine large dense-core vesicles. We characterized chromogranin A and secretogranin II immunoreactivities in cerebrospinal fluid by radioimmunoassays using synthetic peptides derived from these components (GE-25 for chromogranin A and secretoneurin for secretogranin II). In lumbar cerebrospinal fluid, high levels (more than 1000 fmol/ml) of these two components were found, whereas in ventricular cerebrospinal fluid the secretoneurin levels were relatively low. The cerebrospinal fluid/serum ratio for secretoneurin was close to 170. High-performance liquid chromatography revealed that in both cerebrospinal fluid and extracts from human brain secretoneurin was the predominant immunoreactive component. In cerebrospinal fluid chromogranin A immunoreactivity was present as intermediate-sized peptides with little intact chromogranin A and free GE-25 peptide. In human brain samples smaller peptides including GE-25 were more predominant. Analogous findings for secretoneurin and chromogranin A were obtained for bovine brain samples. We can conclude that chromogranins are present in cerebrospinal fluid in concentrations much higher than those of classical neuropeptides also stored in large dense-core vesicles. Therefore, their degree of proteolytic processing can be analysed with small samples of cerebrospinal fluid. A possible disturbance of proteolytic processing in large dense-core vesicles in various pathological conditions can now be discovered.

Chromogranin A immunoreactivity in human cerebrospinal fluid: properties, relationship to noradrenergic neuronal activity, and variation in neurologic disease.

Although measurement of chromogranin A in the bloodstream is of value in sympathoadrenal investigations, little is systematically known about chromogranin A in cerebrospinal fluid, despite substantial knowledge about its occurrence and distribution in brain. We therefore applied a homologous human chromogranin A radioimmunoassay to cerebrospinal fluid, in order to evaluate the properties and stability of cerebrospinal fluid chomogranin A, as well as its relationship to central noradrenergic neuronal activity, to peripheral (plasma) chromogranin A, and to disease states such as hypertension, renal failure and Parkinsonism. Authentic, physically stable chromogranin A immunoreactivity was found in cerebrospinal fluid (at 37-146 ng/ml; mean, 87.0 +/- 6.0 ng/ml in healthy subjects), and several lines of evidence (including 3.39 +/- 0.27-fold higher chromogranin A in cerebrospinal fluid than in plasma) indicated that it originated from a local central nervous system source, rather than the periphery. Cerebrospinal fluid chromogranin A values were not influenced by administration of effective antihypertensive doses of clonidine or propranolol, and were not related to the cerebrospinal fluid concentrations of norepinephrine, methoxyhydroxyphenylglycol, or dopamine-beta-hydroxylase; thus, cerebrospinal fluid chromogranin A was not closely linked to biochemical or pharmacologic indices of central noradrenergic neuronal activity. Cerebrospinal fluid chromogranin A was not changed (P > 0.1) in essential hypertension (84.2 +/- 14.0 ng/ml) or renal failure (72.2 +/- 13.4 ng/ml), despite a marked (7.1-fold; P < 0.001) increase in plasma chromogranin A in renal failure, and a modest (1.5-fold; P = 0.004) increase in plasma chromogranin A in essential hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

CSF chromogranin A-like immunoreactivity in schizophrenia. Assessment of clinical and biochemical relationships.

Chromogranin A (CgA) is co-released with catecholamines and peptides and has a wide distribution in the brain. Chromogranin A provides a measure of tonic arousal. CSF CgA-like immunoreactivity (CgA-LI) was studied in 42 drug-free male schizophrenic patients. 33 of these patients were first studied during chronic haloperidol maintenance treatment. Withdrawal from haloperidol maintenance treatment was associated with a significant increase in CSF CgA-LI, particularly in the patients who did not relapse. Contrary to expectation CSF CgA-LI was higher in drug-free patients who slept longer the night before the lumbar puncture. Significant relationships were observed between CSF CgA-LI and CSF homovanillic acid, acetylcholinesterase, neuropeptide Y-LI and 5-hydroxy-indole acetic acid, but not with CSF norepinephrine or 3-methoxy-4-hydroxyphenylglycol. Ventricular brain ratios correlated negatively with CSF CgA-LI levels.

Reduction of chromogranin A and B but not C in the cerebrospinal fluid in subjects with schizophrenia.

The granins (secretogranins/chromogranins) are a family of soluble proteins stored and released from the secretory large dense-core vesicles of the synapse. Schizophrenia is a common and devastating brain disorder. Although the aetiology of schizophrenia is unknown, data are accumulating that synaptic disturbance or damage may be of importance. The objective of this study was to compare the levels of chromogranin A, B and C in the cerebrospinal fluid (CSF) of patients with schizophrenia and healthy controls. CSF chromogranin levels were measured by RIA in 33 subsequent admissions of patients with psychotic disorder and in 31 healthy controls. The levels of CSF chromogranin A (11.8+/-3.0 vs 14.8+/-4.8 nmol/l, P=0.004), chromogranin B (3.4+/-0.49 vs 3.7+/-0.58 nmol/l, P=0.02), but not chromogranin C (70.2+/-15.7 vs 65.3+/-20.4 pmol/l, P=0.29) were lower in the schizophrenic patients than in the healthy controls. These data indicate that two widespread constituents of large dense-core vesicles, i.e. chromogranin A and chromogranin B, are altered in chronic schizophrenic patients.

Migraine, but not subarachnoid hemorrhage, is associated with differentially increased NPY-like immunoreactivity in the CSF.

To test whether migraine and subarachnoid hemorrhage (SAH) are associated with increased sympathetic tone, we compared the neuropeptide Y-like (NPY-LI) and chromogranin A-like immunoreactivities (LI) of cerebrospinal fluid (CSF) from migraneurs and SAH patients with those from control subjects. Increased sympathetic tone was expected to produce higher co-release of these co-stored peptides and concordant changes in their CSF levels. In addition, we investigated a possible disturbed nitric oxide homeostasis by measuring CSF nitrites (NO). More than 70% of CSF NPY-LI corresponded to the chromatographic peak (HPLC) for the intact molecule in all three groups. Migraneurs had 64% higher CSF NPY-LI, but no significant difference in CSF chromogranin A-LI, as compared to controls. In contrast, SAH patients had 74% less CSF chromogranin A-LI and a trend to lower NPY-LI, as compared to controls. No differences in CSF NO were detected among groups. These results argue against an increased sympathetic tone in patients with either migraine or SAH, and suggest that the higher CSF NPY-LI of migraneurs probably originates from central neurons. Furthermore, our findings in SAH patients argue in favor of a decreased sympathetic tone; this could be a homeostatic response to counterbalance vasoconstriction mediated by other mechanisms.

CSF chromogranin A-like immunoreactivity in schizophrenia: relationships with REM latency and slow wave sleep.

Chromogranin A (CgA) is a calcium binding protein and a precursor of modulatory peptides in the brain. We measured CgA-like immunoreactivity (CgA-LI) in cerebrospinal fluid (CSF) in 15 male schizophrenic patients (diagnosed by DSM-III-R criteria) after 3 nights of polysomnography. Patients had been drug free for at least 33 days. Our earlier report that CSF CgA-LI in schizophrenic patients correlated significantly with negative symptoms and ventricle-brain ratios, which have been related to slow wave sleep, raised the possibility that CgA-LI might relate to slow wave sleep. CSF CgA-LI was significantly correlated with stage 4 sleep and rapid eye movement latency. Whether these positive relationships between CSF CgA-LI and electroencephalographic sleep measures are specific for schizophrenia awaits further study.

Granins as disease-biomarkers: translational potential for psychiatric and neurological disorders.

The identification of biomarkers represents a fundamental medical advance that can lead to an improved understanding of disease pathogenesis, and holds the potential to define surrogate diagnostic and prognostic endpoints. Because of the inherent difficulties in assessing brain function in patients and objectively identifying neurological and cognitive/emotional symptoms, future application of biomarkers to neurological and psychiatric disorders is extremely desirable. This article discusses the biomarker potential of the granin family, a group of acidic proteins present in the secretory granules of a wide variety of endocrine, neuronal and neuroendocrine cells: chromogranin A (CgA), CgB, Secretogranin II (SgII), SgIII, HISL-19 antigen, 7B2, NESP55, VGF and ProSAAS. Their relative abundance, functional significance, and secretion into the cerebrospinal fluid (CSF), saliva, and the general circulation have made granins tractable targets as biomarkers for many diseases of neuronal and endocrine origin, recently impacting diagnosis of a number of neurological and psychiatric disorders including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia, and schizophrenia. Although research has not yet validated the clinical utility of granins as surrogate endpoints for the progression or treatment of neurological or psychiatric disease, a growing body of experimental evidence indicates that the use of granins as biomarkers might be of great potential clinical interest. Advances that further elucidate the mechanism(s) of action of granins, coupled with improvements in biomarker technology and direct clinical application, should increase the translational effectiveness of this family of proteins in disease diagnosis and drug discovery.

Converging pathways of chromogranin and amyloid metabolism in the brain.

Much is unknown regarding the regulation of Alzheimer-related amyloid-beta protein precursor (AbetaPP)-processing in the human central nervous system. It has been hypothesized that amyloidogenic AbetaPP-processing preferentially occurs in the regulated secretory pathway of neurons. To test this hypothesis we looked for correlations of AbetaPP-derived molecules in cerebrospinal fluid (CSF) with chromogranin (Cg) derived peptides, representing the regulated secretion. Patients with Alzheimer's disease (AD, N=32), multiple sclerosis (MS, N=50), and healthy controls (N= 70) were enrolled. CSF was analyzed for the amyloid peptides Abeta1-42, Abetax-42, Abetax-40, Abetax-38, alpha-cleaved soluble AbetaPP (sAbetaPPalpha), beta-cleaved soluble AbetaPP (sAbetaPPbeta), and peptides derived from CgB and SgII (Secretogranin-II, CgC). We investigated CSF levels of the protease BACE1, which processes AbetaPP into Abeta, in relation to Cg-levels. Finally, we measured Cg levels in cell media from untreated and BACE1-inhibited SH-SY5Y human neuroblastoma cells. CSF Cg levels correlated to sAbetaPP and Abeta peptides in AD, MS, and controls, and to CSF BACE1. Cell medium from BACE1-inhibited cells had decreased CgB levels. These results suggest that a large part of AbetaPP in the human central nervous system is processed in the regulated secretory pathway of neurons.

Clinical improvement in parkinsonian patients undergoing adrenal to caudate transplantation is not reflected by chromogranin A or basic fibroblast growth factor in ventricular fluid.

Fifteen patients with Parkinson's disease underwent open transplantation of autologous adrenal medulla to the caudate nucleus. Motor function was evaluated before and after surgery and was found to be significantly improved at 5-9 months following surgery. Cerebrospinal fluid was taken from the ventricle adjacent to the implant site at the beginning of the operation and at 1 week, 3 months, and 5-9 months following surgery. The cerebrospinal fluid was assayed for chromogranin A (CgA), the major soluble protein in chromaffin granules, and basic fibroblast growth factor (bFGF), a neurotrophic growth factor found in normal brain and adrenal medulla. CgA levels did not increase following surgery, suggesting that a significant number of chromaffin cells did not survive or that surviving chromaffin cells did not secrete a significant amount of CgA. Basic fibroblast growth factor was undetectable in the ventricular cerebrospinal fluid.

Levels and proteolytic processing of chromogranin A and B and secretogranin II in cerebrospinal fluid in neurological diseases.

Human cerebrospinal fluid (CSF) contains chromogranin A and B and secretogranin II which represent peptides secreted from neuronal large dense core vesicles. Within these vesicles these precursor peptides are at least partly processed to smaller peptides. We analysed the CSF levels of chromogranins/secretogranin by radioimmunoassay using specific antisera. The degree of their processing was characterized by molecular sieve column chromatography followed by radioimmunoassay. As previously shown secretogranin II is fully processed to smaller peptides including the peptide secretoneurin, whereas processing of chromogranin A was more limited. For chromogranin B we found in this study a high degree of processing comparable to that of secretogranin II. An analysis of CSF from patients with multiple sclerosis, essential tremor, Alzheimer and Parkinson disease, did not reveal any differences in proteolytic processing of chromogranins/secretogranin when compared to control CSF. We conclude that in the four diseases investigated there is no change in the proteolytic processing of the chromogranins/secretogranin within the large dense core vesicles. The absolute levels of chromogranins/secretogranin varied in CSF collected in different hospitals, however their relative ratios were remarkable constant. We suggest to use this ratio as a parameter to standardise CSF levels of other peptides, e.g. neuropeptides. In Parkinson patients the chromogranin A/secretogranin II ratio was significantly increased whereas in Alzheimer patients and those with essential tremor and multiple sclerosis no change of the ratios was observed. Apparently there are only limited changes in the biosynthesis, processing, secretion and CSF clearance of these peptides in pathological conditions.

Chromogranin A in cerebrospinal fluid: a biochemical marker for synaptic degeneration in Alzheimer's disease?

Biochemical markers for AD would be of great value both to improve the clinical diagnostic accuracy in scientific studies and to increase the knowledge of the pathogenesis of the disorder. One of the main features of AD is a degeneration of synapses. Therefore, we examined if chromogranin A (CrA), the major protein of large dense-core synaptic vesicles, in cerebrospinal fluid (CSF) may be of value as a biochemical marker for the synaptic function in AD. The mean concentration of CrA in CSF was about 7.5 times higher than its concentration in serum, and there was no significant correlation between CSF-CrA and the blood-brain barrier function (measured as the CSF/serum albumin ratio), nor between CSF-CrA and serum-CrA. These findings suggest that the major portion of CSF-CrA is locally produced within the CNS. There were no significant differences in CSF-CrA between the AD (n = 29), vascular dementia (n = 13), and age-matched control (n = 9) groups (99.9 +/- 58.9 ng/ml, 108.0 +/- 69.4 ng/ml, and 115.1 +/- 44.4 ng/ml, respectively). However, when the AD group was subdivided into AD type I (n = 12) and AD type II (n = 17), a lower concentration of CSF-CrA was found in AD type I (72.8 +/- 28.9 ng/ml) compared with controls (115.1 +/- 44.4 ng/ml), p < 0.02, and compared with AD type II (119.1 +/- 67.5 ng/ml), p < 0.05, while CSF-CrA did not significantly differ between AD type II and controls. These findings suggest that CSF-CrA has a potential as a biochemical marker for the synaptic degeneration in AD type I, and gives further support for the relevance of identifying the AD type I (pure AD) subgroup in scientific studies.