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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.


Related in: MedlinePlus

Western blots of P2, P3, S3, and S1 fractions based on antibodies against 13 different proteins. This blot composite is representative of four samples for the top seven images and of two samples for the remaining six proteins, all selected randomly from sample #s 4, 6-12, 14-16 (Table 1). Molecular weights from relevant standards are indicated on the right. For all 13 reactivities, the signal was enriched in P3 fractions. FINC: fibronectin, SNAP-23: synaptosomal-associated protein 23, PGHS-2: prostaglandin H synthase, RALA: ras-related protein Ral-A, S5A2: 3-oxo-5-alpha-steroid 4-dehydrogenase, ARL2: ADP-ribosylation factor-like protein 2, SCG3: secretogranin 3.
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Figure 7: Western blots of P2, P3, S3, and S1 fractions based on antibodies against 13 different proteins. This blot composite is representative of four samples for the top seven images and of two samples for the remaining six proteins, all selected randomly from sample #s 4, 6-12, 14-16 (Table 1). Molecular weights from relevant standards are indicated on the right. For all 13 reactivities, the signal was enriched in P3 fractions. FINC: fibronectin, SNAP-23: synaptosomal-associated protein 23, PGHS-2: prostaglandin H synthase, RALA: ras-related protein Ral-A, S5A2: 3-oxo-5-alpha-steroid 4-dehydrogenase, ARL2: ADP-ribosylation factor-like protein 2, SCG3: secretogranin 3.

Mentions: To explore the sources of these structures, we considered that the abundant spherical structures would come from the choroid plexuses and the ependymal lining of the CSF, and might be derived from similar-sized synaptic vesicles [29], LDCVs [30], or exosomes [31], while the blob- and strand-like structures may be derived from extracellular matrix proteins [32]. Nerve terminals known to border the CSF [1] could discharge synaptic vesicles and LDCVs into the CSF. Neighboring cells could also discharge exosomes. While such processes have not been defined for CSF, exosomes have been reported in urine [14], blood [33], and brain extracellular fluid [34]. Accordingly, we performed Western blot analysis on CSF fractions from sample #s 4, 6-12, 14-16, using antibodies against proteins we have identified by LCMS. We used fractions from four different participants for each antibody in 7/13 studies and two participants for the other 6 antibodies, chosen randomly from the same 11 different participants, as for electrophoresis. We found 13 proteins are enriched in P3s (Figure 7). Seven proteins are significantly enriched in P3 (n = 3 or 4, p < 0.05, paired analysis), based on specific staining at their predicted molecular weight: FINC, SNAP 23, PGHS-2, RALA, S5A2, ARL2, and SCG3. We also found that semaphorin 4D, chromogranin A & B, and the known SNARE complex proteins synaptotagmin, syntaxin, and synaptobrevin were enriched in P3, but with insufficient samples for statistical analysis.


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)

Western blots of P2, P3, S3, and S1 fractions based on antibodies against 13 different proteins. This blot composite is representative of four samples for the top seven images and of two samples for the remaining six proteins, all selected randomly from sample #s 4, 6-12, 14-16 (Table 1). Molecular weights from relevant standards are indicated on the right. For all 13 reactivities, the signal was enriched in P3 fractions. FINC: fibronectin, SNAP-23: synaptosomal-associated protein 23, PGHS-2: prostaglandin H synthase, RALA: ras-related protein Ral-A, S5A2: 3-oxo-5-alpha-steroid 4-dehydrogenase, ARL2: ADP-ribosylation factor-like protein 2, SCG3: secretogranin 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2746175&req=5

Figure 7: Western blots of P2, P3, S3, and S1 fractions based on antibodies against 13 different proteins. This blot composite is representative of four samples for the top seven images and of two samples for the remaining six proteins, all selected randomly from sample #s 4, 6-12, 14-16 (Table 1). Molecular weights from relevant standards are indicated on the right. For all 13 reactivities, the signal was enriched in P3 fractions. FINC: fibronectin, SNAP-23: synaptosomal-associated protein 23, PGHS-2: prostaglandin H synthase, RALA: ras-related protein Ral-A, S5A2: 3-oxo-5-alpha-steroid 4-dehydrogenase, ARL2: ADP-ribosylation factor-like protein 2, SCG3: secretogranin 3.
Mentions: To explore the sources of these structures, we considered that the abundant spherical structures would come from the choroid plexuses and the ependymal lining of the CSF, and might be derived from similar-sized synaptic vesicles [29], LDCVs [30], or exosomes [31], while the blob- and strand-like structures may be derived from extracellular matrix proteins [32]. Nerve terminals known to border the CSF [1] could discharge synaptic vesicles and LDCVs into the CSF. Neighboring cells could also discharge exosomes. While such processes have not been defined for CSF, exosomes have been reported in urine [14], blood [33], and brain extracellular fluid [34]. Accordingly, we performed Western blot analysis on CSF fractions from sample #s 4, 6-12, 14-16, using antibodies against proteins we have identified by LCMS. We used fractions from four different participants for each antibody in 7/13 studies and two participants for the other 6 antibodies, chosen randomly from the same 11 different participants, as for electrophoresis. We found 13 proteins are enriched in P3s (Figure 7). Seven proteins are significantly enriched in P3 (n = 3 or 4, p < 0.05, paired analysis), based on specific staining at their predicted molecular weight: FINC, SNAP 23, PGHS-2, RALA, S5A2, ARL2, and SCG3. We also found that semaphorin 4D, chromogranin A & B, and the known SNARE complex proteins synaptotagmin, syntaxin, and synaptobrevin were enriched in P3, but with insufficient samples for statistical analysis.

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