Traumatic brain injury enhances neuroinflammation and lesion volume in caveolin deficient mice

BACKGROUND: Traumatic brain injury (TBI) enhances pro-inflammatory responses, neuronal loss and long-term behavioral deficits. Caveolins (Cavs) are regulators of neuronal and glial survival signaling. Previously we showed that astrocyte and microglial activation is increased in Cav-1 knock-out (KO) mice and that Cav-1 and Cav-3 modulate microglial morphology. We hypothesized that Cavs may regulate cytokine production after TBI. METHODS: Controlled cortical impact (CCI) model of TBI (3 m/second; 1.0 mm depth; parietal cortex) was performed on wild-type (WT; C57Bl/6), Cav-1 KO, and Cav-3 KO mice. Histology and immunofluorescence microscopy (lesion volume, glia activation), behavioral tests (open field, balance beam, wire grip, T-maze), electrophysiology, electron paramagnetic resonance, membrane fractionation, and multiplex assays were performed. Data were analyzed by unpaired t tests or analysis of variance (ANOVA) with post-hoc Bonferroni’s multiple comparison. RESULTS: CCI increased cortical and hippocampal injury and decreased expression of MLR-localized synaptic proteins (24 hours), enhanced NADPH oxidase (Nox) activity (24 hours and 1 week), enhanced polysynaptic responses (1 week), and caused hippocampal-dependent learning deficits (3 months). CCI increased brain lesion volume in both Cav-3 and Cav-1 KO mice after 24 hours (P < 0.0001, n = 4; one-way ANOVA). Multiplex array revealed a significant increase in expression of IL-1β, IL-9, IL-10, KC (keratinocyte chemoattractant), and monocyte chemoattractant protein 1 (MCP-1) in ipsilateral hemisphere and IL-9, IL-10, IL-17, and macrophage inflammatory protein 1 alpha (MIP-1α) in contralateral hemisphere of WT mice after 4 hours. CCI increased IL-2, IL-6, KC and MCP-1 in ipsilateral and IL-6, IL-9, IL-17 and KC in contralateral hemispheres in Cav-1 KO and increased all 10 cytokines/chemokines in both hemispheres except for IL-17 (ipsilateral) and MIP-1α (contralateral) in Cav-3 KO (versus WT CCI). Cav-3 KO CCI showed increased IL-1β, IL-9, KC, MCP-1, MIP-1α, and granulocyte-macrophage colony-stimulating factor in ipsilateral and IL-1β, IL-2, IL-9, IL-10, and IL-17 in contralateral hemispheres (P = 0.0005, n = 6; two-way ANOVA) compared to Cav-1 KO CCI. CONCLUSION: CCI caused astrocyte and microglial activation and hippocampal neuronal injury. Cav-1 and Cav-3 KO exhibited enhanced lesion volume and cytokine/chemokine production after CCI. These findings suggest that Cav isoforms may regulate neuroinflammatory responses and neuroprotection following TBI

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

The Role of Caveolin-1 in Type 2 Diabetes-Induced Alzheimer's Disease

Type 2 Diabetes (T2D) is a risk factor for the development of late onset Alzheimer’s disease. However, this mechanism underlying induction of AD in T2D is largely unknown. Here we show that increased pro-inflammatory cytokines in diabetic mouse models db/db (Leprdb) and MKR (Ckm-IGF1RK1003R), results in depletion of the endothelial-enriched protein Caveolin-1 (Cav-1). In turn, reduced Cav-1 in the hippocampus of db/db mice causes a decrease in the expression level of insulin receptor leading to reduced, insulin transport into the brains of diabetic mice. Further, depletion of Cav-1 induces the upregulation of amyloid precursor protein (APP) and its amyloidogenic cleavage, as well as upregulation in levels of hyperphosphorylated tau. Moreover, db/db and MKR mice exhibit deficits in recognition memory and hippocampal neurogenesis. Furthermore, our results suggest that Cav-1 is essential for neural progenitor cell proliferation and maintenance in the adult brain and its depletion may compromise neurogenesis via bone morphogenetic protein (BMP) signaling. Taken together, these results suggest that loss of Cav-1 levels is a key step in the escalation of T2D into AD

Presenilin-1 Dependent Neurogenesis Regulates Hippocampal Learning and Memory

<div><p>Presenilin-1 (PS1), the catalytic core of the aspartyl protease γ-secretase, regulates adult neurogenesis. However, it is not clear whether the role of neurogenesis in hippocampal learning and memory is PS1-dependent, or whether PS1 loss of function in adult hippocampal neurogenesis can cause learning and memory deficits. Here we show that downregulation of PS1 in hippocampal neural progenitor cells causes progressive deficits in pattern separation and novelty exploration. New granule neurons expressing reduced PS1 levels exhibit decreased dendritic branching and dendritic spines. Further, they exhibit reduced survival. Lastly, we show that PS1 effect on neurogenesis is mediated via β-catenin phosphorylation and notch signaling. Together, these observations suggest that impairments in adult neurogenesis induce learning and memory deficits and may play a role in the cognitive deficits observed in Alzheimer’s disease.</p></div

Compromised notch-1 cleavage and premature cell differentiation in neural progenitor cells expressing reduced levels of PS1 can be partially rescued by exogenous NICD.

<p><b>A</b>. Reduced expression of PS1-NTF, NICD (80 & 120 kDa fragments) and PDGFRα in neural progenitor cells that were infected with lentiviral vectors expressing PS1 shRNA compared to cells infected with control RL shRNA. <b>B<i>i</i>-B<i>ii</i></b>. Densitometry analysis of Western blots. <b>C</b>. Scheme of infection of lentiviral vectors followed by NICD transfection. <b>D, F</b>. Phase contrast images of neural progenitor cells infected with lentiviral vectors expressing either control RL shRNA (D) or PS1 shRNA (F) followed by transfection with either control- or NICD-expressing vector. <b>E, G</b>. Quantification of round cells following NICD transfection show no change in the control condition (<b>E)</b> and a significant increase in the PS1 condition <b>(G)</b> indicating a reversal in morphological change following transfection with NICD-expressing vector with cells exhibiting round morphology (unpaired t-test; *P<0.05). Error bars indicate ±SEM.</p

Compromised survival of new neurons expressing reduced levels of PS1 in the granular cell layer of the dentate gyrus of adult mice.

<p>Unbiased stereological analysis of green fluorescent protein positive (GFP⁺) cell populations in the SGL and GCL of the DG at 3 and 6 months post-injection. <b>A</b>. Less mature neurons within the GCL (GFP⁺BrdU^-NeuN⁺; *P<0.05) of mice injected with PS1 shRNA. <b>B</b>. Separate Comparisons within RL and PS1 groups at 3 and 6 months post injection reveals reduced survival of new neurons in the GCL of PS1 shRNA (GFP⁺BrdU⁺NeuN⁺; *P<0.05). Error bars indicate ±SEM. <b>C</b>. Confocal image (Zeiss LSM 510) representing colocalization of GFP with NeuN and BrdU (63x). <b>(D,E)</b>. Comparisons within RL and PS1 groups at 3 and 6 months post injection shows reduced rate of survival of PDGFRα⁺ NPCs in the SGL (GFP⁺PDGFRα⁺Nestin^-; **P<0.01) <b>(D)</b> and of total neurons and oligodendrocytes within the SGL (GFP⁺PDGFRα⁺; **P<0.01), <b>(E)</b>. Error bars indicate ±SEM. <b>F</b>. Confocal image (Zeiss LSM 510) representing colocalization of GFP, nestin and PDGFRα (63x).</p

PS1 downregulation in new neurons decreases dendritic arborization and spine density.

<p><b>A</b>. Compressed Z-stack confocal images (Zeiss LSM 510) of newly mature GFP⁺ neurons (GFP⁺BrdU+NeuN⁺) in the granular cell layer of the dentate gyrus of RL shRNA- and PS1 shRNA- injected mice 6 months after injection. Images show a dramatic decrease in the number of dendritic arborization in mature GFP⁺ neurons in the PS1 shRNA- injected mice. <b>B</b>. Sholl analysis of the number of intersections measured from n = 16 neurons per group. PS1 animals show decreased number of intersections (two-way ANOVA (treatment and distance from soma) F_{(treatment)1,120} = 14.75, P<0.001, F_{(distance)19,120} = 4.10, P<0.0001). <b>C, D</b>. PS1 downregulation results in decreased number of spines <b>(C)</b> as a function of increasing distance from the soma (two-way ANOVA (treatment and distance from soma) F_{(treatment)1,60} = 13.10, P<0.001, F_{(distance)19,60} = 3.18, P<0.001) and mean spine density <b>(D)</b> (unpaired t-test, *P<0.05). <b>F, G</b>. Dendritic surface area (<b>F)</b> and volume <b>(G)</b> were unaffected by PS1 downregulation. Error bars indicate ±SEM.</p