Plasma-type gelsolin is decreased in human blood and cerebrospinal fluid after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) pathophysiology involves neurovascular proteolysis and inflammation. How these 2 phenomena are related remains unclear. We hypothesize that matrix metalloproteinases (MMPs) mediate the depletion of anti-inflammatory plasma-type gelsolin (pGSN). METHODS: We enrolled 42 consecutive SAH subjects and sampled cerebrospinal fluid (CSF) and blood on post-SAH Days 2 to 3, 4 to 5, 6 to 7, and 10 to 14. Control subjects were 20 consecutive non-SAH hydrocephalus patients with lumbar drains. Enzyme-linked immunosorbent assay, Western blotting, and zymography were used to quantify pGSN and MMP-9. RESULTS: In CSF, pGSN was lower in SAH compared with control subjects on post-SAH Days 2 to 3 (P=0.0007), 4 to 5 (P=0.041), and 10 to 14 (P=0.007). In blood, pGSN decreased over time (P=0.001) and was lower in SAH compared with control subjects on post-SAH Days 4 to 5 (P=0.037), 6 to 7 (P=0.006), and 10 to 14 (P=0.006). Western blots demonstrated that SAH CSF had novel bands at 52 and 46 kDa, representing cleaved pGSN fragments. Gelatin zymography showed that CSF MMP-9 was elevated in SAH compared with control subjects. Higher CSF MMP-9 correlated with lower CSF pGSN on post-SAH Day 7 (r=-0.38; P=0.05). CONCLUSIONS: SAH is associated with decreased CSF and blood pGSN and elevated CSF MMP-9. Novel cleaved pGSN fragments are present in CSF of SAH subjects, consistent with pGSN cleavage by MMPs. Because pGSN is known to inhibit inflammatory mediators, these findings suggest that MMPs may reduce pGSN and exacerbate inflammation after SAH. Further studies are warranted to investigate the mechanisms underlying MMP-pGSN signaling in SAH.

Gelsolin in cerebrospinal fluid as a potential biomarker of epilepsy.

Gelsolin is an actin regulatory protein that generally distributed in a wide variety of body tissues, especially the brain tissues and cerebrospinal fluid. In this study we found that lumbar CSF-gelsolin concentrations markedly decreased in epileptic patients by enzyme linked immunosorbent assay. In order to help judge the result, we determined gelsolin expression in temporal lobe tissues of patients with temporal lobe epilepsy using double-label immunofluorescence to location and using western blot to quantitation. Then we observed that gelsolin was co-expressed with microtubule-associated protein-2 in axons and cytoplasms of neurons and gelsolin protein level was also down-regulated in temporal lobe tissues of epileptic patients. Our findings suggested that CSF-gelsolin level might reflect the alteration of gelsolin in brain tissue of epileptic patients and CSF-gelsolin seems to be a potential biomarker for epilepsy.

Plasma gelsolin modulates cellular response to sphingosine 1-phosphate.

Hypogelsolinemia is observed in patients with different states of acute or chronic inflammation such as sepsis, rheumatoid arthritis, and multiple sclerosis. In animal models of sepsis, repletion of plasma gelsolin reduces septic mortality. However, the functions of extracellular gelsolin and the mechanisms leading to its protective nature are poorly understood. Potential mechanisms involve gelsolin's extracellular actin scavenging function or its ability to bind bioactive lipids or proinflammatory mediators, which would limit inflammatory responses and prevent tissue damage. Here we report that human plasma gelsolin binds to sphingosine 1-phosphate (S1P), a pleiotropic cellular agonist involved in various immune responses, and to its synthetic structural analog FTY720P (Gilenya). The fluorescence intensity of a rhodamine B-labeled phosphatidylinositol 4,5-bisphosphate binding peptide derived from gelsolin and the optical density of recombinant human plasma gelsolin (rhpGSN) were found to decrease after the addition of S1P or FTY720P. Gelsolin's ability to depolymerize F-actin also decreased progressively with increasing addition of S1P. Transient increases in phosphorylation of extracellular signal-regulated kinase in bovine aortic endothelial cells (BAECs) after S1P treatment were inhibited by rhpGSN. The ability of S1P to increase F-actin content and the elastic modulus of primary astrocytes and BAECs was also prevented by rhpGSN. Evaluation of S1P and gelsolin levels in cerebrospinal fluid reveals a low concentration of gelsolin and a high concentration of S1P in samples obtained from patients suffering from lymphatic meningitis. These findings suggest that gelsolin-mediated regulation of S1P bioactivity may be important to maintain immunomodulatory balance at inflammatory sites.

Immunoreactivity of anti-gelsolin antibodies: implications for biomarker validation.

BACKGROUND: Proteomic-based discovery of biomarkers for disease has recently come under scrutiny for a variety of issues; one prominent issue is the lack of orthogonal validation for biomarkers following discovery. Validation by ELISA or Western blot requires the use of antibodies, which for many potential biomarkers are under-characterized and may lead to misleading or inconclusive results. Gelsolin is one such biomarker candidate in HIV-associated neurocognitive disorders. METHODS: Samples from human (plasma and CSF), monkey (plasma), monocyte-derived macrophage (supernatants), and commercial gelsolin (recombinant and purified) were quantitated using Western blot assay and a variety of anti-gelsolin antibodies. Plasma and CSF was used for immunoaffinity purification of gelsolin which was identified in eight bands by tandem mass spectrometry. RESULTS: Immunoreactivity of gelsolin within samples and between antibodies varied greatly. In several instances, multiple bands were identified (corresponding to different gelsolin forms) by one antibody, but not identified by another. Moreover, in some instances immunoreactivity depended on the source of gelsolin, e.g. plasma or CSF. Additionally, some smaller forms of gelsolin were identified by mass spectrometry but not by any antibody. Recombinant gelsolin was used as reference sample. CONCLUSIONS: Orthogonal validation using specific monoclonal or polyclonal antibodies may reject biomarker candidates from further studies based on misleading or even false quantitation of those proteins, which circulate in various forms in body fluids.

Mass spectrometric characterization of gelsolin isoforms.

Gelsolin is the most widely expressed member of the actin capping and severing family of proteins. There are two isoforms of gelsolin: isoform 1, a secretory (plasma) protein that is 51 amino acids longer than isoform 2, a cytosolic protein, at the N-terminus; the first 27 amino acids is a signal sequence. Both isoforms are coded by a single gene and differ as a result of alternative initiation site/splicing. The level of gelsolin in the blood and cerebrospinal fluid (CSF) is altered in many diseases including amyloidoses and other neurodegenerative disorders. Although quantitative analysis of gelsolin has been reported, lack of suitable antibodies makes it impossible to differentiate these two isoforms by immunodetection techniques and no other technique is available. Therefore, ambiguity exists whether gelsolin present in circulation is isoform 1 or also isoform 2 released from lysed cells. We report in this communication a mass spectrometric approach to identify isoform 1 of gelsolin immunopurified from human plasma and CSF. Recombinant isoform 1 was used as reference.

Proteomics of tumor-specific proteins in cerebrospinal fluid of patients with astrocytoma: usefulness of gelsolin protein.

Changes in cerebrospinal fluid (CSF) composition have been shown to accurately reflect pathological processes in the CNS, and are potential indicators of abnormal CNS states, such as tumor growth. To detect biomarkers in high-grade astrocytomas, the differential expression of proteins in the cerebrospinal fluid was analyzed from two cases each of diffuse astrocytoma (grade II), and glioblastoma (grade IV) using agarose 2-D gel electrophoresis (2-DE). It was found that the expression of gelsolin protein decreased with histological grade. To examine whether gelsolin is a useful indicator of tumor aggressiveness or patient outcome, its expression was further studied on immunohistochemistry in 41 formalin-fixed and paraffin-embedded astrocytomas. The positive cell rate of gelsolin in tumors was 59.4% in grade II, 30.0% in grade III and 29.4% in grade IV, respectively. Gelsolin expression was significantly lower in high-grade astrocytomas (grade III or IV) than in low-grade astrocytomas (grade II; P < 0.05). Moreover, in astrocytomas the overall survival of patients in the low-expression group was significantly poorer than in the high expression group (P < 0.05). These data suggest that gelsolin is a prognostic factor in astrocytoma.

Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease.

So far only the detection of 14-3-3 proteins in cerebrospinal fluid (CSF) is included in the diagnostic criteria for sporadic Creutzfeldt-Jakob disease (sCJD). However, this assay cannot be used for screening because of the high rate of false positive results in sCJD, and often negative results in variant CJD. To facilitate the differential diagnosis of CJD, we applied 2-D differential gel-electrophoresis (2-D DIGE) as a quantitative proteomic screening system for CSF proteins. We compared 36 patients suffering from sCJD with 30 patients suffering from other neurodegenerative diseases. Sample preparation was optimized in consideration of the fact that CSF is composed of blood- and brain-derived proteins, and an improved 2-D DIGE protocol was established. Using this method in combination with protein identification by MALDI-TOF-MS, several known surrogate markers of sCJD like 14-3-3 protein, neuron-specific enolase, and lactate dehydrogenase were readily identified. Moreover, a not yet identified protein with an approximate molecular mass of 85 kDa was found as marker for sCJD with high diagnostic specificity and sensitivity. We conclude that our proteomic approach is useful to differentiate CJD from other neurodegenerative diseases and expect that CSF-optimized 2-D DIGE will find broad application in the search for other brain derived proteins in CSF.

Gelsolin concentration in cerebrospinal fluid from patients with multiple sclerosis and other neurological disorders.

BACKGROUND: Gelsolin is a highly conserved intracellular actin-binding protein with an extracellular isoform, plasma gelsolin, for which there is not yet a clearly defined function. MATERIALS AND METHODS: In this study, we determined gelsolin concentrations in blood and cerebrospinal fluid (CSF) obtained from 25 subjects using immunoblotting and a functional assay that quantifies gelsolin's ability to accelerate actin polymerization. RESULTS: The gelsolin concentration in CSF, determined by quantitative immunoblotting was 1.2-15.9 microg/ml (average 5.9 +/- 3.8 mug/ml). In samples obtained from patients diagnosed with conditions that do not alter standard CSF clinical tests [(idiopathic cephalgia, ischialgia due to discopathy, and idiopathic (Bell's) facial nerve palsy or entrapment radial neuropathy)], the average gelsolin concentration was 7.2 +/- 4.3 microg/ml. In contrast, the gelsolin concentration in samples obtained from patients diagnosed with multiple sclerosis was 2.1 +/- 0.7 microg/ml, and a similar low concentration was found in a patient recovering from a subarachnoid hemorrhage. The range of CSF gelsolin concentrations determined by the actin polymerization assay was 0.61-9.97 microg/ml (average 3.6 +/- 2.2 microg/ml). These lower values compared with those obtained from immunoblotting analysis suggest that CSF gelsolin may bind other CSF molecules leading to a reduction of its actin-binding activity. CONCLUSIONS: The results presented here show that CSF gelsolin concentration is significantly altered in certain neurological conditions, including multiple sclerosis, indicating the possible utility of CSF gelsolin levels for diagnostic purposes.

Neuroprotective effects of sildenafil in experimental spinal cord injury in rabbits.

Neuroprotective agents such as methylprednisolone and sildenafil may limit damage after spinal cord injury. We evaluated the effects of methylprednisolone and sildenafil on biochemical and histologic changes after spinal cord injury in a rabbit model. Female New Zealand rabbits (32 rabbits) were allocated to 4 equal groups: laminectomy only (sham control) or laminectomy and spinal trauma with no other treatment (trauma control) or treatment with either methylprednisolone or sildenafil. Gelsolin and caspase-3 levels in cerebrospinal fluid and plasma were determined, and spinal cord histology was evaluated at 24 hours after trauma. There were no differences in mean cerebrospinal fluid or plasma levels of caspase-3 between the groups or within the groups from 0 to 24 hours after injury. From 0 to 24 hours after trauma, mean cerebrospinal fluid gelsolin levels significantly increased in the sildenafil group and decreased in the sham control and the trauma control groups. Mean plasma gelsolin level was significantly higher at 8 and 24 hours after trauma in the sildenafil than other groups. Histologic examination indicated that general structural integrity was better in the methylprednisolone in comparison with the trauma control group. General structural integrity, leptomeninges, white and grey matter hematomas, and necrosis were significantly improved in the sildenafil compared with the trauma control group. Caspase-3 levels in the cerebrospinal fluid and blood were not increased but gelsolin levels were decreased after spinal cord injury in trauma control rabbits. Sildenafil caused an increase in gelsolin levels and may be more effective than methylprednisolone at decreasing secondary damage to the spinal cord. 

Vitamin D therapy in experimental allergic encephalomyelitis could be limited by opposing effects of sphingosine 1-phosphate and gelsolin dysregulation.

Several studies support a protective effect of vitamin D on multiple sclerosis and experimental allergic encephalomyelitis (EAE), but the mechanisms of these favorable effects are unclear. Our study demonstrates that sphingosine 1-phosphate (S1P) is upregulated in the serum and spinal cords of EAE rats, but that vitamin D reverses the upregulation to alleviate inflammation. Vitamin D, however, cannot prevent the disease process, suggesting that other factors may be involved. To identify additional factors that might limit vitamin D efficacy, we assessed the effects of vitamin D on plasma gelsolin (pGSN), a regulator of S1P that is downregulated in the CSF of MS patients. Our results show that pGSN is downregulated in the serum of EAE rats, whereas its cellular form, cytoplasmic gelsolin (cGSN), is upregulated in the spinal cord of EAE rats. Importantly, vitamin D causes a downregulation of both pGSN and cGSN, which may counteract the positive effects of S1P decrease. Furthermore, 48 and 42 kDa caspase-3 cleavage products of cGSN are detected in EAE spinal cords, suggesting enhanced apoptotic activity, but these cleaved products undergo a similar decrease upon vitamin D treatment. To directly test the role of cGSN in the apoptotic process, we performed RNA interference in PC-12, a rat sympathetic nerve cell line. Results verify that cGSN suppresses apoptosis induced by TNF-α. Collectively, these results support a therapeutic effect of vitamin D that is derived from its ability to reduce S1P, but is limited by its simultaneous effect in reducing pGSN and cGSN. Based on these observations, we postulate that combined therapy with recombinant human pGSN and vitamin D may produce more beneficial effect in treating multiple sclerosis.