Limits...
Transcriptome analysis of synaptoneurosomes identifies neuroplasticity genes overexpressed in incipient Alzheimer's disease.

Williams C, Mehrian Shai R, Wu Y, Hsu YH, Sitzer T, Spann B, McCleary C, Mo Y, Miller CA - PLoS ONE (2009)

Bottom Line: These patients also showed increased expression of neuroplasticity related genes, many encoding 3'UTR consensus sequences that regulate translation in the synapse.An increase in mRNA encoding the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) was paralleled by elevated expression of the corresponding protein in IAD.These results imply a functional impact on synaptic transmission as GluR2, if inserted, maintains the receptors in a low conductance state.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Keck School of Medicine University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
In Alzheimer's disease (AD), early deficits in learning and memory are a consequence of synaptic modification induced by toxic beta-amyloid oligomers (oAbeta). To identify immediate molecular targets downstream of oAbeta binding, we prepared synaptoneurosomes from prefrontal cortex of control and incipient AD (IAD) patients, and isolated mRNAs for comparison of gene expression. This novel approach concentrates synaptic mRNA, thereby increasing the ratio of synaptic to somal mRNA and allowing discrimination of expression changes in synaptically localized genes. In IAD patients, global measures of cognition declined with increasing levels of dimeric Abeta (dAbeta). These patients also showed increased expression of neuroplasticity related genes, many encoding 3'UTR consensus sequences that regulate translation in the synapse. An increase in mRNA encoding the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) was paralleled by elevated expression of the corresponding protein in IAD. These results imply a functional impact on synaptic transmission as GluR2, if inserted, maintains the receptors in a low conductance state. Some overexpressed genes may induce early deficits in cognition and others compensatory mechanisms, providing targets for intervention to moderate the response to dAbeta.

Show MeSH

Related in: MedlinePlus

Enrichment and stability of synaptoneurosomes.Microscopy: A) Nuclear contamination of the homogenate (2 µl smears) (DAPI fluorescence) decreases after sequential passage through mesh screens with B) the post- 10 µm screen synaptoneurosome pellet (100 µl volume) still retaining a few nuclei (Bar = 20 µm). C) Intact synaptoneurosomes are detected by phase contrast in smears of pelleted post-10 µm screen synaptoneurosome as typical “snowman” pre- and postsynaptic profiles (arrowheads). Larger, empty, membranous structures are also observed. (Bar = 5 µm). D) Electron microscopy reveal “snowman” profiles with apparent postsynaptic densities (arrowheads); (Bar = 300 nm). E) At higher magnification, presynaptic terminals containing synaptic vesicles, 20 nm in size (white arrows) are indicated (Bar = 100 nm). Immunoblots: F) Homogenates (black bars) and synaptoneurosomes (white bars) were compared for stability and enrichment of synaptic proteins and contamination with other cellular debris. Densitometric comparison on immunoblots of postsynaptic proteins PSD-95, NMDAR1 and GluR2 in synaptoneurosomes shows more than a two-fold enrichment compared to homogenates, an increase in presynaptic protein SNAP-25 but a 40% decrease in glial protein GFAP. Cytoplasmic proteins β-tubulin and PKCα show little change. The ratio of each protein to GAPDH is given in arbitrary units based on the integrated density value which is the sum of all pixel values after background correction (IDV). G) In synaptoneurosomes, de novo synthesis of several proteins is observed as newly translated, biotinylated proteins detected with streptavidin (lane 1), and one at 50 kD co-migrates with a band detected with antibody to αCAMKII (lane 4,arrowhead). Actinomycin D, a transcription inhibitor, does not reduce the protein band profile (lane 2). Protein synthesis is inhibited by the translation inhibitor, anisomycin (lane 3), although endogenously biotinylated proteins present in synaptoneurosomes are still seen at ∼140 and 75 kD (asterisks).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2654156&req=5

pone-0004936-g001: Enrichment and stability of synaptoneurosomes.Microscopy: A) Nuclear contamination of the homogenate (2 µl smears) (DAPI fluorescence) decreases after sequential passage through mesh screens with B) the post- 10 µm screen synaptoneurosome pellet (100 µl volume) still retaining a few nuclei (Bar = 20 µm). C) Intact synaptoneurosomes are detected by phase contrast in smears of pelleted post-10 µm screen synaptoneurosome as typical “snowman” pre- and postsynaptic profiles (arrowheads). Larger, empty, membranous structures are also observed. (Bar = 5 µm). D) Electron microscopy reveal “snowman” profiles with apparent postsynaptic densities (arrowheads); (Bar = 300 nm). E) At higher magnification, presynaptic terminals containing synaptic vesicles, 20 nm in size (white arrows) are indicated (Bar = 100 nm). Immunoblots: F) Homogenates (black bars) and synaptoneurosomes (white bars) were compared for stability and enrichment of synaptic proteins and contamination with other cellular debris. Densitometric comparison on immunoblots of postsynaptic proteins PSD-95, NMDAR1 and GluR2 in synaptoneurosomes shows more than a two-fold enrichment compared to homogenates, an increase in presynaptic protein SNAP-25 but a 40% decrease in glial protein GFAP. Cytoplasmic proteins β-tubulin and PKCα show little change. The ratio of each protein to GAPDH is given in arbitrary units based on the integrated density value which is the sum of all pixel values after background correction (IDV). G) In synaptoneurosomes, de novo synthesis of several proteins is observed as newly translated, biotinylated proteins detected with streptavidin (lane 1), and one at 50 kD co-migrates with a band detected with antibody to αCAMKII (lane 4,arrowhead). Actinomycin D, a transcription inhibitor, does not reduce the protein band profile (lane 2). Protein synthesis is inhibited by the translation inhibitor, anisomycin (lane 3), although endogenously biotinylated proteins present in synaptoneurosomes are still seen at ∼140 and 75 kD (asterisks).

Mentions: To augment studies comparing homogenates of control to AD brains, we used synaptoneurosome preparations [35] to enrich synaptic mRNAs. Synaptoneurosomes were prepared from the prefrontal cortices of control and IAD patients by a simple, rapid, but gentle method using sequential mesh screens. Preparations were analyzed for residual nuclei by applying 4′-6-Diamidino-2-phenylindole (DAPI)-containing mounting media to smears of each pellet. The percentage of nuclei in the synaptoneurosome pellet was reduced to 0.4% of those in the homogenate (Figure 1A, 1B) and are, therefore, a minor source for contaminating mRNAs. Most nuclei were observed densely packed in the first pellet (P1, not shown).


Transcriptome analysis of synaptoneurosomes identifies neuroplasticity genes overexpressed in incipient Alzheimer's disease.

Williams C, Mehrian Shai R, Wu Y, Hsu YH, Sitzer T, Spann B, McCleary C, Mo Y, Miller CA - PLoS ONE (2009)

Enrichment and stability of synaptoneurosomes.Microscopy: A) Nuclear contamination of the homogenate (2 µl smears) (DAPI fluorescence) decreases after sequential passage through mesh screens with B) the post- 10 µm screen synaptoneurosome pellet (100 µl volume) still retaining a few nuclei (Bar = 20 µm). C) Intact synaptoneurosomes are detected by phase contrast in smears of pelleted post-10 µm screen synaptoneurosome as typical “snowman” pre- and postsynaptic profiles (arrowheads). Larger, empty, membranous structures are also observed. (Bar = 5 µm). D) Electron microscopy reveal “snowman” profiles with apparent postsynaptic densities (arrowheads); (Bar = 300 nm). E) At higher magnification, presynaptic terminals containing synaptic vesicles, 20 nm in size (white arrows) are indicated (Bar = 100 nm). Immunoblots: F) Homogenates (black bars) and synaptoneurosomes (white bars) were compared for stability and enrichment of synaptic proteins and contamination with other cellular debris. Densitometric comparison on immunoblots of postsynaptic proteins PSD-95, NMDAR1 and GluR2 in synaptoneurosomes shows more than a two-fold enrichment compared to homogenates, an increase in presynaptic protein SNAP-25 but a 40% decrease in glial protein GFAP. Cytoplasmic proteins β-tubulin and PKCα show little change. The ratio of each protein to GAPDH is given in arbitrary units based on the integrated density value which is the sum of all pixel values after background correction (IDV). G) In synaptoneurosomes, de novo synthesis of several proteins is observed as newly translated, biotinylated proteins detected with streptavidin (lane 1), and one at 50 kD co-migrates with a band detected with antibody to αCAMKII (lane 4,arrowhead). Actinomycin D, a transcription inhibitor, does not reduce the protein band profile (lane 2). Protein synthesis is inhibited by the translation inhibitor, anisomycin (lane 3), although endogenously biotinylated proteins present in synaptoneurosomes are still seen at ∼140 and 75 kD (asterisks).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654156&req=5

pone-0004936-g001: Enrichment and stability of synaptoneurosomes.Microscopy: A) Nuclear contamination of the homogenate (2 µl smears) (DAPI fluorescence) decreases after sequential passage through mesh screens with B) the post- 10 µm screen synaptoneurosome pellet (100 µl volume) still retaining a few nuclei (Bar = 20 µm). C) Intact synaptoneurosomes are detected by phase contrast in smears of pelleted post-10 µm screen synaptoneurosome as typical “snowman” pre- and postsynaptic profiles (arrowheads). Larger, empty, membranous structures are also observed. (Bar = 5 µm). D) Electron microscopy reveal “snowman” profiles with apparent postsynaptic densities (arrowheads); (Bar = 300 nm). E) At higher magnification, presynaptic terminals containing synaptic vesicles, 20 nm in size (white arrows) are indicated (Bar = 100 nm). Immunoblots: F) Homogenates (black bars) and synaptoneurosomes (white bars) were compared for stability and enrichment of synaptic proteins and contamination with other cellular debris. Densitometric comparison on immunoblots of postsynaptic proteins PSD-95, NMDAR1 and GluR2 in synaptoneurosomes shows more than a two-fold enrichment compared to homogenates, an increase in presynaptic protein SNAP-25 but a 40% decrease in glial protein GFAP. Cytoplasmic proteins β-tubulin and PKCα show little change. The ratio of each protein to GAPDH is given in arbitrary units based on the integrated density value which is the sum of all pixel values after background correction (IDV). G) In synaptoneurosomes, de novo synthesis of several proteins is observed as newly translated, biotinylated proteins detected with streptavidin (lane 1), and one at 50 kD co-migrates with a band detected with antibody to αCAMKII (lane 4,arrowhead). Actinomycin D, a transcription inhibitor, does not reduce the protein band profile (lane 2). Protein synthesis is inhibited by the translation inhibitor, anisomycin (lane 3), although endogenously biotinylated proteins present in synaptoneurosomes are still seen at ∼140 and 75 kD (asterisks).
Mentions: To augment studies comparing homogenates of control to AD brains, we used synaptoneurosome preparations [35] to enrich synaptic mRNAs. Synaptoneurosomes were prepared from the prefrontal cortices of control and IAD patients by a simple, rapid, but gentle method using sequential mesh screens. Preparations were analyzed for residual nuclei by applying 4′-6-Diamidino-2-phenylindole (DAPI)-containing mounting media to smears of each pellet. The percentage of nuclei in the synaptoneurosome pellet was reduced to 0.4% of those in the homogenate (Figure 1A, 1B) and are, therefore, a minor source for contaminating mRNAs. Most nuclei were observed densely packed in the first pellet (P1, not shown).

Bottom Line: These patients also showed increased expression of neuroplasticity related genes, many encoding 3'UTR consensus sequences that regulate translation in the synapse.An increase in mRNA encoding the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) was paralleled by elevated expression of the corresponding protein in IAD.These results imply a functional impact on synaptic transmission as GluR2, if inserted, maintains the receptors in a low conductance state.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Keck School of Medicine University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
In Alzheimer's disease (AD), early deficits in learning and memory are a consequence of synaptic modification induced by toxic beta-amyloid oligomers (oAbeta). To identify immediate molecular targets downstream of oAbeta binding, we prepared synaptoneurosomes from prefrontal cortex of control and incipient AD (IAD) patients, and isolated mRNAs for comparison of gene expression. This novel approach concentrates synaptic mRNA, thereby increasing the ratio of synaptic to somal mRNA and allowing discrimination of expression changes in synaptically localized genes. In IAD patients, global measures of cognition declined with increasing levels of dimeric Abeta (dAbeta). These patients also showed increased expression of neuroplasticity related genes, many encoding 3'UTR consensus sequences that regulate translation in the synapse. An increase in mRNA encoding the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) was paralleled by elevated expression of the corresponding protein in IAD. These results imply a functional impact on synaptic transmission as GluR2, if inserted, maintains the receptors in a low conductance state. Some overexpressed genes may induce early deficits in cognition and others compensatory mechanisms, providing targets for intervention to moderate the response to dAbeta.

Show MeSH
Related in: MedlinePlus