Loss of Caveolin-1 Accelerates Neurodegeneration and Aging

The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimer's. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimer's disease.We analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aβ, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aβ expression.Therefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimer's disease

Ischemic preconditioning (IPC) does not occur in Cav-1 KO mice.

<p>(<b>A</b>) Hippocampal synaptosomes from Cav-1 KO (Yg) showed a similar pattern to Ag, with a decrease in PSD-95, NR2A, NR2B, and AMPAR. PSD-95 IPs of Cav-1 KO synaptosomes revealed minimal detection in PSD-95, NR2A, NR2B, and AMPAR. (<b>B</b>) WT or Cav-1 KO mice were subjected to 3 min (ischemic preconditioning, IPC) and/or 12 min (lethal ischemia, LI) induced by bilateral carotid artery occlusion (BCAO). Intact neurons in CA1 hippocampal (HP) region were counted from Cresyl Violet stained paraffin fixed sections. IPC (3 min, BCAO) significantly protected CA1 neurons against LI (12 min, BCAO) in WT mice (<b>iv</b>). There was a significant increase in CA1 neuronal death in Cav-1 KO animals subject to IPC (<b>viii</b>) versus WT IPC + LI. Representative Cresyl Violet stained CA1 hippocampal images from (<b>i</b>) WT sham, (<b>ii</b>) WT IPC, (<b>iii</b>) WT LI, and (<b>iv</b>) WT IPC and (<b>v</b>) Cav-1 KO sham, (<b>vi</b>) Cav-1 KO IPC, (<b>vii</b>) Cav-1 KO LI, and (<b>viii</b>) Cav-1 KO IPC. Quantitation of images is presented by the graph.</p

Cav-1 KO mice exhibit enhanced astrogliosis and neuronal degeneration.

<p>(<b>A–C</b>) Light microscopic image displaying 0.5 µm thick hippocampal sections of Cav-1 KO (<b>A</b><i>-i, </i><b>A</b><i>-ii</i>), aged (<b>B</b><i>-i, </i><b>B</b><i>-ii</i>), and young (<b>C</b><i>-i, </i><b>C</b><i>-ii</i>) stained with toludine blue. There is a drastic reduction in neurons within the dentate gyrus (large arrow heads) and CA1 regions (arrows) of young Cav-1 KO mice compared to young and aged WT. In addition, there appears to be the presence of more glia and glial scar formation within the dentate gyrus of Cav-1 KO mice as indicated by the darker gray cell bodies intermixed with the neurons. (<b>D</b>) Hippocampal coronal cryostat sections (10 µm) from WT and Cav-1 KO mice were stained with Nissl (neuronal marker, red pixels) and GFAP (astrocyte marker, green) to show no overlap between neurons and astrocytes. (<b>E</b>) Coronal cryostat sections (25 µm) of 2 month WT, 2 month Cav-1 KO and 12 month Cav-1 KO stained with 0.0004% <i>Flouro-Jade®B</i> and fluorescent red Nissl with DAPI. Areas from CA1 of the hippocampus were imaged. WT CA1 showed well-organized astrocytes. Two month Cav-1 KO had areas of disorganized astrocytes with lightly labeling areas of potential future plaque development. Twelve month Cav-1 KO CA1 areas had large bright, entangled green fluorescence with red neurons inside and significantly less organized astrocytes, further demonstrating a degenerating neuronal model.</p

Hippocampal homogenates show an aged dependent reduction in NR2A, NR2B, PSD-95, and Cav-1.

<p>Hippocampi were isolated from the brains of C57BL/6J mice at 3–6 months (young, Yg), 12 months (middle aged, Md), and 24 months (aged, Ag). Immunoblot and densitometric analysis demonstrated a significant reduction in PSD-95, NR2A, NR2B, TrkBR, and Cav-1 in the Md and Ag hippocampus compared to Yg.</p

PSD-95, NR2A, NR2B, AMPAR, TrkBR, and Cav-1 are abundantly detected in buoyant fractions (BF) from young mouse brains homogenates, yet are less abundant BFs from middle aged and aged brains.

<p>Sucrose density fractionated was performed on brains from three different age groups of C57BL/6J mice: young (Yg, 3–6 months), middle aged (Md, 12 months), and aged (Ag, >18 months). Immunoblot analysis detected the majority of PSD-95 (post-synaptic density marker), NR2A, NR2B, AMPAR, TrkBR, and Cav-1 in buoyant fractions 4 and 5 (BFs) isolated from Yg brains (<b>A</b>). In contrast, the Md and Ag brains exhibited a drastic reduction in these synaptic signaling components, with the majority of these proteins detected in heavy fractions 11 and 12 (HFs) only. Densitometric analysis of the data is represented in <b>B</b>. (<b>C</b>) Cav-1 (<b><i>C</i></b>) and PSD-95 (<b><i>P</i></b>) immunoprecipitates pulled down NR2A, NR2B, AMPAR, and TrkB in the buoyant fractions of Yg mice, with decreased detection in Md and Ag. (<b>D</b>) Immunoblot analysis detected a significant decrease in PSD-95 (post-synaptic density marker), NR2A, NR2B, AMPAR, and Cav-1 in hippocampal synaptosomes from Md and Ag brains compared to Yg. PSD-95, NR2, NR2B, AMPAR, and Cav-1 decreased in PSD-95 immunoprecipitates of Md, and Ag synaptosomes compared to Yg. (<b>E</b>) Electron paramagnetic resonance (EPR) was performed on synaptosomal membranes from brains of C57BL/6J mice: young (Yg, 3–6 months) and aged (Old, >18 months). Membrane localized spin labels 5-doxylstearic acid (5-DSA) probes changes in the neuronal membrane fluidity closer to the membrane surface. Lineshape analysis of 5-DSA spin label using the indicated parameters revealed that neuronal membrane of aged mice exhibit significantly lower order parameter (i.e. increased fluidity) than young animals. Aged membranes were 8.5±1.2% more fluid than young membranes (F_(1,10) = 223.5, p = 0).</p

Cav-1 KO mice have reduced hippocampal synapses.

<p>Synapses were quantified by routine electron microscopy as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015697#pone.0015697-Head2" target="_blank">[82]</a>. EM analysis revealed a significant reduction in hippocampal synapses in both (<b>C</b>) Cav-1 KO (Yg) and (<b>B</b>) Ag mice compared to (<b>A</b>) WT. Synapses are indicated by red circles in WT, blue circles in Ag, and green circles in Cav-1 KO. (<b>D</b>) WT micrographs exhibited dendritic processes (indicated by d) with intact cytoskeletal architecture (arrows and arrowheads), while (<b>E</b>) Ag and (<b>F</b>) Cav-1 KO displayed less organized dendritic shafts (asterisk) with more abundant astrocyte presence (arrows). (<b>G</b>) Quantitation of data.</p

Neuron-targeted re-expression of Cav-1 reduces Aβ expression in primary neurons cultured from Cav-1 KO brains.

<p>Primary neurons from Cav-1 KO mice were grown in culture for 4 days and transfected a lentiviral vector containing Cav-1 driven by the synapsin promoter (<i>SynCav1</i>) for 72 hr. <i>SynGFP</i> served as control vector (<i>2×</i>10⁹ viral particles for both vectors). Schematic of the vector is shown in <b>A</b>. Increasing doses of <i>SynCav1</i> proportionally decreased Aβ expression (<b>B</b>). Six separate primary cultures of Cav-1 KO neurons were incubated with either <i>SynGFP</i> or <i>SynCav1</i>. <i>SynCav1</i> significantly decreased Aβ expression after 72 hr.</p