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The morphology and biochemistry of nanostructures provide evidence for synthesis and signaling functions in human cerebrospinal fluid.

Harrington MG, Fonteh AN, Oborina E, Liao P, Cowan RP, McComb G, Chavez JN, Rush J, Biringer RG, Hühmer AF - Cerebrospinal Fluid Res (2009)

Bottom Line: Nanostructure fractions had a unique composition compared to CSF supernatant, richer in omega-3 and phosphoinositide lipids, active prostanoid enzymes, and fibronectin.Unique morphology and biochemistry features of abundant and discrete membrane-bound CSF nanostructures are described.Prostaglandin H synthase activity, essential for prostanoid production and previously unknown in CSF, is localized to nanospheres.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Neurology, Huntington Medical Research Institutes, Pasadena, CA, 91101 USA. mghworks@hmri.org.

ABSTRACT

Background: Cerebrospinal fluid (CSF) contacts many brain regions and may mediate humoral signaling distinct from synaptic neurotransmission. However, synthesis and transport mechanisms for such signaling are not defined. The purpose of this study was to investigate whether human CSF contains discrete structures that may enable the regulation of humoral transmission.

Methods: Lumbar CSF was collected prospectively from 17 participants: with no neurological or psychiatric disease, with Alzheimer's disease, multiple sclerosis, or migraine; and ventricular CSF from two cognitively healthy participants with long-standing shunts for congenital hydrocephalus. Cell-free CSF was subjected to ultracentrifugation to yield supernatants and pellets that were examined by transmission electron microscopy, shotgun protein sequencing, electrophoresis, western blotting, lipid analysis, enzymatic activity assay, and immuno-electron microscopy.

Results: Over 3,600 CSF proteins were identified from repeated shotgun sequencing of cell-free CSF from two individuals with Alzheimer's disease: 25% of these proteins are normally present in membranes. Abundant nanometer-scaled structures were observed in ultracentrifuged pellets of CSF from all 16 participants examined. The most common structures included synaptic vesicle and exosome components in 30-200 nm spheres and irregular blobs. Much less abundant nanostructures were present that derived from cellular debris. Nanostructure fractions had a unique composition compared to CSF supernatant, richer in omega-3 and phosphoinositide lipids, active prostanoid enzymes, and fibronectin.

Conclusion: Unique morphology and biochemistry features of abundant and discrete membrane-bound CSF nanostructures are described. Prostaglandin H synthase activity, essential for prostanoid production and previously unknown in CSF, is localized to nanospheres. Considering CSF bulk flow and its circulatory dynamics, we propose that these nanostructures provide signaling mechanisms via volume transmission within the nervous system that are for slower, more diffuse, and of longer duration than synaptic transmission.

No MeSH data available.


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Prostanoid regulation in CSF. A: prostaglandin H synthase (PGHS-1 and PGHS-2) activity assays for P3, S3, and S1 from two different study participants, analyzed for a total of at least three measures per fraction, with standard error bars. Both PGHS-1 & PGHS-2 specific activities are demonstrated in P3 and S1 versus S3 fractions, compared to baseline activities without inhibitor. This demonstrates the presence of specific PGHS-1 and -2 activities in the S1 fractions that are enriched in the P3 (and decreased in the S3) fractions. B: Scheme for prostanoid enzymes, receptors, and regulators identified in CSF by shotgun liquid chromatography mass spectrometry (blue) and substrates identified by LCMS in SRM mode (green). Prostaglandins were not identified in this study (black). This diagram outlines CSF components capable of extensive prostanoid synthesis, with receptors and regulators, including the functional enzymes PGHS-1 and -2 (Figure 10A), the critical source of prostaglandin H2 (PGH2). PLA2: phospholipase A2; PTGDS: prostaglandin D synthase; PGES: prostaglandin E synthase; PGIS: prostaglandin I synthase; THAS: thromboxin A synthase; PGD: prostaglandin D; PGE: prostaglandin E; PGI: prostaglandin I; TXA: thromboxane A; PD2R: prostaglandin D2 receptor; PE2R1, 2, 3, 4: prostaglandin E1, 2, 3, 4 receptors; PI2R: prostaglandin I2 receptor; TA2R: thromboxane A2 receptor; FEM1A: Prostaglandin E receptor 4-associated protein.
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Figure 10: Prostanoid regulation in CSF. A: prostaglandin H synthase (PGHS-1 and PGHS-2) activity assays for P3, S3, and S1 from two different study participants, analyzed for a total of at least three measures per fraction, with standard error bars. Both PGHS-1 & PGHS-2 specific activities are demonstrated in P3 and S1 versus S3 fractions, compared to baseline activities without inhibitor. This demonstrates the presence of specific PGHS-1 and -2 activities in the S1 fractions that are enriched in the P3 (and decreased in the S3) fractions. B: Scheme for prostanoid enzymes, receptors, and regulators identified in CSF by shotgun liquid chromatography mass spectrometry (blue) and substrates identified by LCMS in SRM mode (green). Prostaglandins were not identified in this study (black). This diagram outlines CSF components capable of extensive prostanoid synthesis, with receptors and regulators, including the functional enzymes PGHS-1 and -2 (Figure 10A), the critical source of prostaglandin H2 (PGH2). PLA2: phospholipase A2; PTGDS: prostaglandin D synthase; PGES: prostaglandin E synthase; PGIS: prostaglandin I synthase; THAS: thromboxin A synthase; PGD: prostaglandin D; PGE: prostaglandin E; PGI: prostaglandin I; TXA: thromboxane A; PD2R: prostaglandin D2 receptor; PE2R1, 2, 3, 4: prostaglandin E1, 2, 3, 4 receptors; PI2R: prostaglandin I2 receptor; TA2R: thromboxane A2 receptor; FEM1A: Prostaglandin E receptor 4-associated protein.

Mentions: We wanted to test PGHS activity from a large quantity of CSF because PGHS-2 staining structures were infrequent on iTEM (Figure 8A) and P3 western blots for PGHS-2 were faint (Figure 7). Accordingly, we used 90 mL of ventricular CSF from two patients with congenital hydrocephalus from sample #s 18 & 19 (both clinically stable, with normal pressure and total protein, and free from infection). We tested fractions from each person separately and in duplicate and found activity in S1 and P3 that was reduced or absent in S3; inhibitor studies reveal that the activity was from both PGHS-1 and PGHS-2 (Figure 10A).


The morphology and biochemistry of nanostructures provide evidence for synthesis and signaling functions in human cerebrospinal fluid.

Harrington MG, Fonteh AN, Oborina E, Liao P, Cowan RP, McComb G, Chavez JN, Rush J, Biringer RG, Hühmer AF - Cerebrospinal Fluid Res (2009)

Prostanoid regulation in CSF. A: prostaglandin H synthase (PGHS-1 and PGHS-2) activity assays for P3, S3, and S1 from two different study participants, analyzed for a total of at least three measures per fraction, with standard error bars. Both PGHS-1 & PGHS-2 specific activities are demonstrated in P3 and S1 versus S3 fractions, compared to baseline activities without inhibitor. This demonstrates the presence of specific PGHS-1 and -2 activities in the S1 fractions that are enriched in the P3 (and decreased in the S3) fractions. B: Scheme for prostanoid enzymes, receptors, and regulators identified in CSF by shotgun liquid chromatography mass spectrometry (blue) and substrates identified by LCMS in SRM mode (green). Prostaglandins were not identified in this study (black). This diagram outlines CSF components capable of extensive prostanoid synthesis, with receptors and regulators, including the functional enzymes PGHS-1 and -2 (Figure 10A), the critical source of prostaglandin H2 (PGH2). PLA2: phospholipase A2; PTGDS: prostaglandin D synthase; PGES: prostaglandin E synthase; PGIS: prostaglandin I synthase; THAS: thromboxin A synthase; PGD: prostaglandin D; PGE: prostaglandin E; PGI: prostaglandin I; TXA: thromboxane A; PD2R: prostaglandin D2 receptor; PE2R1, 2, 3, 4: prostaglandin E1, 2, 3, 4 receptors; PI2R: prostaglandin I2 receptor; TA2R: thromboxane A2 receptor; FEM1A: Prostaglandin E receptor 4-associated protein.
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Figure 10: Prostanoid regulation in CSF. A: prostaglandin H synthase (PGHS-1 and PGHS-2) activity assays for P3, S3, and S1 from two different study participants, analyzed for a total of at least three measures per fraction, with standard error bars. Both PGHS-1 & PGHS-2 specific activities are demonstrated in P3 and S1 versus S3 fractions, compared to baseline activities without inhibitor. This demonstrates the presence of specific PGHS-1 and -2 activities in the S1 fractions that are enriched in the P3 (and decreased in the S3) fractions. B: Scheme for prostanoid enzymes, receptors, and regulators identified in CSF by shotgun liquid chromatography mass spectrometry (blue) and substrates identified by LCMS in SRM mode (green). Prostaglandins were not identified in this study (black). This diagram outlines CSF components capable of extensive prostanoid synthesis, with receptors and regulators, including the functional enzymes PGHS-1 and -2 (Figure 10A), the critical source of prostaglandin H2 (PGH2). PLA2: phospholipase A2; PTGDS: prostaglandin D synthase; PGES: prostaglandin E synthase; PGIS: prostaglandin I synthase; THAS: thromboxin A synthase; PGD: prostaglandin D; PGE: prostaglandin E; PGI: prostaglandin I; TXA: thromboxane A; PD2R: prostaglandin D2 receptor; PE2R1, 2, 3, 4: prostaglandin E1, 2, 3, 4 receptors; PI2R: prostaglandin I2 receptor; TA2R: thromboxane A2 receptor; FEM1A: Prostaglandin E receptor 4-associated protein.
Mentions: We wanted to test PGHS activity from a large quantity of CSF because PGHS-2 staining structures were infrequent on iTEM (Figure 8A) and P3 western blots for PGHS-2 were faint (Figure 7). Accordingly, we used 90 mL of ventricular CSF from two patients with congenital hydrocephalus from sample #s 18 & 19 (both clinically stable, with normal pressure and total protein, and free from infection). We tested fractions from each person separately and in duplicate and found activity in S1 and P3 that was reduced or absent in S3; inhibitor studies reveal that the activity was from both PGHS-1 and PGHS-2 (Figure 10A).

Bottom Line: Nanostructure fractions had a unique composition compared to CSF supernatant, richer in omega-3 and phosphoinositide lipids, active prostanoid enzymes, and fibronectin.Unique morphology and biochemistry features of abundant and discrete membrane-bound CSF nanostructures are described.Prostaglandin H synthase activity, essential for prostanoid production and previously unknown in CSF, is localized to nanospheres.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Neurology, Huntington Medical Research Institutes, Pasadena, CA, 91101 USA. mghworks@hmri.org.

ABSTRACT

Background: Cerebrospinal fluid (CSF) contacts many brain regions and may mediate humoral signaling distinct from synaptic neurotransmission. However, synthesis and transport mechanisms for such signaling are not defined. The purpose of this study was to investigate whether human CSF contains discrete structures that may enable the regulation of humoral transmission.

Methods: Lumbar CSF was collected prospectively from 17 participants: with no neurological or psychiatric disease, with Alzheimer's disease, multiple sclerosis, or migraine; and ventricular CSF from two cognitively healthy participants with long-standing shunts for congenital hydrocephalus. Cell-free CSF was subjected to ultracentrifugation to yield supernatants and pellets that were examined by transmission electron microscopy, shotgun protein sequencing, electrophoresis, western blotting, lipid analysis, enzymatic activity assay, and immuno-electron microscopy.

Results: Over 3,600 CSF proteins were identified from repeated shotgun sequencing of cell-free CSF from two individuals with Alzheimer's disease: 25% of these proteins are normally present in membranes. Abundant nanometer-scaled structures were observed in ultracentrifuged pellets of CSF from all 16 participants examined. The most common structures included synaptic vesicle and exosome components in 30-200 nm spheres and irregular blobs. Much less abundant nanostructures were present that derived from cellular debris. Nanostructure fractions had a unique composition compared to CSF supernatant, richer in omega-3 and phosphoinositide lipids, active prostanoid enzymes, and fibronectin.

Conclusion: Unique morphology and biochemistry features of abundant and discrete membrane-bound CSF nanostructures are described. Prostaglandin H synthase activity, essential for prostanoid production and previously unknown in CSF, is localized to nanospheres. Considering CSF bulk flow and its circulatory dynamics, we propose that these nanostructures provide signaling mechanisms via volume transmission within the nervous system that are for slower, more diffuse, and of longer duration than synaptic transmission.

No MeSH data available.


Related in: MedlinePlus