Chronic Rapamycin Restores Brain Vascular Integrity and Function Through NO Synthase Activation and Improves Memory in Symptomatic Mice Modeling Alzheimer’s Disease

Vascular pathology is a major feature of Alzheimer's disease (AD) and other dementias. We recently showed that chronic administration of the target-of-rapamycin (TOR) inhibitor rapamycin, which extends lifespan and delays aging, halts the progression of AD-like disease in transgenic human (h)APP mice modeling AD when administered before disease onset. Here we demonstrate that chronic reduction of TOR activity by rapamycin treatment started after disease onset restored cerebral blood flow (CBF) and brain vascular density, reduced cerebral amyloid angiopathy and microhemorrhages, decreased amyloid burden, and improved cognitive function in symptomatic hAPP (AD) mice. Like acetylcholine (ACh), a potent vasodilator, acute rapamycin treatment induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO release in brain endothelium. Administration of the NOS inhibitor L-NG-Nitroarginine methyl ester reversed vasodilation as well as the protective effects of rapamycin on CBF and vasculature integrity, indicating that rapamycin preserves vascular density and CBF in AD mouse brains through NOS activation. Taken together, our data suggest that chronic reduction of TOR activity by rapamycin blocked the progression of AD-like cognitive and histopathological deficits by preserving brain vascular integrity and function. Drugs that inhibit the TOR pathway may have promise as a therapy for AD and possibly for vascular dementias

Relapse prevention for addictive behaviors

The Relapse Prevention (RP) model has been a mainstay of addictions theory and treatment since its introduction three decades ago. This paper provides an overview and update of RP for addictive behaviors with a focus on developments over the last decade (2000-2010). Major treatment outcome studies and meta-analyses are summarized, as are selected empirical findings relevant to the tenets of the RP model. Notable advances in RP in the last decade include the introduction of a reformulated cognitive-behavioral model of relapse, the application of advanced statistical methods to model relapse in large randomized trials, and the development of mindfulness-based relapse prevention. We also review the emergent literature on genetic correlates of relapse following pharmacological and behavioral treatments. The continued influence of RP is evidenced by its integration in most cognitive-behavioral substance use interventions. However, the tendency to subsume RP within other treatment modalities has posed a barrier to systematic evaluation of the RP model. Overall, RP remains an influential cognitive-behavioral framework that can inform both theoretical and clinical approaches to understanding and facilitating behavior change

Immunoproteasome Deficiency Protects in the Retina after Optic Nerve Crush

<div><p>The immunoproteasome is upregulated by disease, oxidative stress, and inflammatory cytokines, suggesting an expanded role for the immunoproteasome in stress signaling that goes beyond its canonical role in generating peptides for antigen presentation. The signaling pathways that are regulated by the immunoproteasome remain elusive. However, previous studies suggest a role for the immunoproteasome in the regulation of PTEN and NF-κB signaling. One well-known pathway upstream of NF-κB and downstream of PTEN is the Akt signaling pathway, which is responsible for mediating cellular survival and is modulated after optic nerve crush (ONC). This study investigated the role of retinal immunoproteasome after injury induced by ONC, focusing on the Akt cell survival pathway. Retinas or retinal pigment epithelial (RPE) cells from wild type (WT) and knockout (KO) mice lacking either one (LMP2) or two (LMP7 and MECL-1) catalytic subunits of the immunoproteasome were utilized in this study. We show that mRNA and protein levels of the immunoproteasome subunits are significantly upregulated in WT retinas following ONC. Mice lacking the immunoproteasome subunits show either a delayed or dampened apoptotic response as well as altered Akt signaling, compared to WT mice after ONC. Treatment of the RPE cells with insulin growth factor-1 (IGF-1) to stimulate Akt signaling confirmed that the immunoproteasome modulates this pathway, and most likely modulates parallel pathways as well. This study links the inducible expression of the immunoproteasome following retinal injury to Akt signaling, which is important in many disease pathways.</p></div

Immunoproteasome Deficiency Results in Altered Ganglion Cell Death after ONC.

<p>(<b>A, <i>left</i></b>) Representative retinal section 11 days following ONC stained with DAPI (blue) and TUNEL (green). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Image was taken with a 20x objective and the scale bar indicates 50 μm. (<b>A, <i>right</i></b>) Representative retinal whole mount stained with anti-βIII-tubulin (green), TUNEL (red, arrow), and DAPI (blue). Image was taken with 100x objective and the scale bar indicates 10 μm. (7 days following ONC) (<b>B</b>) Summary of the percent TUNEL-positive cells in the GCL following ONC from WT (●), L2 (■), and L7M1 (▲) mice. Results are the mean ± S.E. of 3–9 mice/group compared to control (day 0). (*, p≤0.01 (WT) and #, p≤0.05 (L2) by one-way ANOVA with Dunnett’s post-test compared to the ‘day 0’ values). (<b>C</b>) Summary of GCL nuclei density (nuclei/μM) following ONC from WT (●), L2 (■), and L7M1 (▲) mice. Results are the mean ± S.E. of 3–9 mice/group compared to control (day 0). (*, p≤0.01 (WT) and ^, p≤0.05 (L7M1) by one-way ANOVA with Dunnett’s post-test compared to the ‘day 0’ values).</p

Immunoproteasome Modulates RPE Cell Signaling after IGF-1 Stimulation.

<p>RPE cells isolated from WT, L2, and L7M1 mice were serum starved for 24h and treated with IGF (100ng/mL) for the indicated time points. Results are the optical density of PTEN, p-Akt/Akt, and p-S6K1/S6K1 protein content. Data are shown as a proportion of each mouse strains’ control (day 0) and represent the mean ± S.E. of 3–6 repeats. (*, p≤0.05 by one-way ANOVA with Dunnett’s post-test compared to the ‘No Drug’ values; ^, p≤0.05 by one-way ANOVA with Dunnett’s post-test compared to the ‘30 min’ values.</p

Protein Content in WT, L2, and L7M1 Murine Retinas before Injury.

<p>The optical density of protein content from uncrushed WT, L2, and L7M1 mouse retinas normalized to WT mice. Protein content is shown for PTEN, p-Akt/Akt, Akt, p-S6K1, S6K1, Bcl-2, and Bax. Results represent the mean ± S.E. of 4–9 mice/group. (*, p≤0.001 by one-way ANOVA with Dunnett’s post-test compared to WT mouse retina).</p

Immunoproteasome Deficiency Alters Content of Stress Proteins after ONC.

<p>(<b>A</b> and <b>B</b>) Protein was isolated from WT, L2, or L7M1 retinas at the indicated time points after ONC. Results are the optical density of PTEN, p-Akt/Akt, and p-S6K1/S6K1 (<b>A</b>), or Bcl-2 and Bax (<b>B</b>) protein content. Data is shown relative to each mouse strains’ control (day 0) and represent the mean ± S.E. of 3–9 mice/group. (*, p≤0.05 by one-way ANOVA with Dunnett’s post-test compared to the ‘day 0’ values or (^, p≤0.05 by one-way ANOVA with Dunnett’s post-test compared to the ‘day 2’ values).</p

Mononuclear cell response in mouse retina post-ONC.

<p>Recruitment of CD45+ cells to retina in CD11c-DTR/GFP mice on the B6 background. Retinas were harvested from naive mice, and from mice at intervals ranging from 1 day to 10 wks post-ONC. (<b>A</b>) Dendritic cells (DC) were identified as CD45+CD11b+Ly6G-GFP+. (<b>B</b>) Microglia/macrophages (MG/Mac) were identified CD45+CD11b+Ly6G-GFP-. (<b>C</b>) Neutrophils (PMN) were CD45+CD11b+Ly6G+GFP-. Other immune cells were present in very small numbers, and not significantly changed by the ONC. Closed bars—ipsilateral (ONC-treated) retinas; open bars—contralateral control retinas. Results are the mean ± S.D. of 4–5 mice/group.</p

Optic Nerve Crush Increases Stress and Correlates with an Increase in Immunoproteasome Subunits in WT Retina.

<p>(<b>A</b> and <b>B</b>) Protein was isolated from WT (●), L2 (□), and L7M1 (Δ) mouse retinas at the indicated time points after ONC. Results are the optical density of immunoproteasome subunits (<b>A</b>) LMP7 (β5i) and (<b>B</b>) LMP2 (β1i), and are the mean ± S.E. of 3–22** mice/group compared to control (day 0). (*, p≤0.05 (WT), and #, p≤0.01 (L2) by one-way ANOVA with Dunnett’s post-test compared to the ‘day 0’ values). (** All 36 day WT mouse data represents 2 mice/group). (<b>C</b>) WT retina tissue sections stained with anti-GFAP in WT retina before and after optic nerve crush (11 days). Image was taken with a 40x objective. (<b>D</b>) Protein was isolated from WT (●), L2 (■), and L7M1 (▲) retinas at the indicated time points after ONC. Results are the optical density of the GFAP protein content, and are the mean ± S.E. of 3–15 mice/group compared to control (day 0) for each mouse strain. (*, p≤0.001 (WT), #, p≤0.01 (L2) by one-way ANOVA with Dunnett’s post-test compared to the ‘Day 0’ values). (<b>E</b>) GFAP and LMP7 protein content isolated from WT (●) or L2 (□) mice are shown as a correlation. (R² = 0.2292; p≤0.01 (WT), R² = 0.1620; p≤0.01 (L2), by Pearson Correlation). (<b>F</b>) GFAP and LMP2 protein content from WT (●) or (Δ) L7M1 mice are shown as a correlation. (R² = 0.2860; p≤0.001 (WT), R² = 0.0126, p = 0.57 (L7M1), by Pearson Correlation).</p

Antibodies used for Immunohistochemistry or Western Immunoblotting.

<p>Monoclonal (M), polyclonal (P), Immunohistochemistry or Immunofluorescence (I), Western immunoblotting (W).</p><p>Antibodies used for Immunohistochemistry or Western Immunoblotting.</p