Inhibition of the classical pathway of the complement cascade prevents early dendritic and synaptic degeneration in glaucoma

BACKGROUND: Glaucoma is a complex, multifactorial disease characterised by the loss of retinal ganglion cells and their axons leading to a decrease in visual function. The earliest events that damage retinal ganglion cells in glaucoma are currently unknown. Retinal ganglion cell death appears to be compartmentalised, with soma, dendrite and axon changes potentially occurring through different mechanisms. There is mounting evidence from other neurodegenerative diseases suggesting that neuronal dendrites undergo a prolonged period of atrophy, including the pruning of synapses, prior to cell loss. In addition, recent evidence has shown the role of the complement cascade in synaptic pruning in glaucoma and other diseases. RESULTS: Using a genetic (DBA/2J mouse) and an inducible (rat microbead) model of glaucoma we first demonstrate that there is loss of retinal ganglion cell synapses and dendrites at time points that precede axon or soma loss. We next determine the role of complement component 1 (C1) in early synaptic loss and dendritic atrophy during glaucoma. Using a genetic knockout of C1qa (D2.C1qa (-/-) mouse) or pharmacological inhibition of C1 (in the rat bead model) we show that inhibition of C1 is sufficient to preserve dendritic and synaptic architecture. CONCLUSIONS: This study further supports assessing the potential for complement-modulating therapeutics for the prevention of retinal ganglion cell degeneration in glaucoma. Mol Neurodegener 2016 Apr 6; 11(2):2

Meox2 Haploinsufficiency Accelerates Axonal Degeneration in DBA/2J Glaucoma.

Purpose: Glaucoma is a complex disease with major risk factors including advancing age and increased intraocular pressure (IOP). Dissecting these earliest events will likely identify new avenues for therapeutics. Previously, we performed transcriptional profiling in DBA/2J (D2) mice, a widely used mouse model relevant to glaucoma. Here, we use these data to identify and test regulators of early gene expression changes in DBA/2J glaucoma. Methods: Upstream regulator analysis (URA) in Ingenuity Pathway Analysis was performed to identify potential master regulators of differentially expressed genes. The function of one putative regulator, mesenchyme homeobox 2 (Meox2), was tested using a combination of genetic, biochemical, and immunofluorescence approaches. Results: URA identified Meox2 as a potential regulator of early gene expression changes in the optic nerve head (ONH) of DBA/2J mice. Meox2 haploinsufficiency did not affect the characteristic diseases of the iris or IOP elevation seen in DBA/2J mice but did cause a significant increase in the numbers of eyes with axon damage compared to controls. While young mice appeared normal, aged Meox2 haploinsufficient DBA/2J mice showed a 44% reduction in MEOX2 protein levels. This correlated with modulation of age- and disease-specific vascular and myeloid alterations. Conclusions: Our data support a model whereby Meox2 controls IOP-dependent vascular remodeling and neuroinflammation to promote axon survival. Promoting these earliest responses prior to IOP elevation may be a viable neuroprotective strategy to delay or prevent human glaucoma

DBA/2J Genetic Background Exacerbates Spontaneous Lethal Seizures but Lessens Amyloid Deposition in a Mouse Model of Alzheimer’s Disease

<div><p>Alzheimer’s disease (AD) is a leading cause of dementia in the elderly and is characterized by amyloid plaques, neurofibrillary tangles (NFTs) and neuronal dysfunction. Early onset AD (EOAD) is commonly caused by mutations in amyloid precursor protein (APP) or genes involved in the processing of APP including the presenilins (e.g. PSEN1 or PSEN2). In general, mouse models relevant to EOAD recapitulate amyloidosis, show only limited amounts of NFTs and neuronal cell dysfunction and low but significant levels of seizure susceptibility. To investigate the effect of genetic background on these phenotypes, we generated <i>APP^swe</i> and <i>PSEN1^de9</i> transgenic mice on the seizure prone inbred strain background, DBA/2J. Previous studies show that the DBA/2J genetic background modifies plaque deposition in the presence of mutant APP but the impact of <i>PSEN1^de9</i> has not been tested. Our study shows that DBA/2J.<i>APP^swePSEN1^de9</i> mice are significantly more prone to premature lethality, likely to due to lethal seizures, compared to B6.<i>APP^swePSEN1^de9</i> mice—70% of DBA/2J.<i>APP^swePSEN1^de9</i> mice die between 2-3 months of age. Of the DBA/2J.<i>APP^swePSEN1^de9</i> mice that survived to 6 months of age, plaque deposition was greatly reduced compared to age-matched B6.<i>APP^swePSEN1^de9</i> mice. The reduction in plaque deposition appears to be independent of microglia numbers, reactive astrocytosis and complement C5 activity.</p></div

Microglia respond equally in D2.<i>APB</i>^<i>Tg</i> and B6.<i>APB</i>^<i>Tg</i> mice compared to wild type controls.

<p>(<b>A-D</b>) Representative images of IBA1⁺ cells in the cortex of B6 (A), B6.<i>APB</i>^<i>Tg</i> (B), D2 (C) and D2.<i>APB</i>^<i>Tg</i> (D) mice. (<b>E</b>) IBA1⁺ cells were counted in three different regions of the cortex in all 4 strains of mice. The levels of microglia show a mild increase in D2.<i>APB</i>^<i>Tg</i> and B6.<i>APB</i>^<i>Tg</i> mice compared to their wild type controls mice, but there is no significant difference microglia numbers comparing D2.<i>APB</i>^<i>Tg</i> with B6.<i>APB</i>^<i>Tg</i> mice. NS = not significant. Scale Bar = 100μm.</p

No overt difference in glial responses in plaque regions in D2.<i>APB</i>^<i>Tg</i> compared to B6.<i>APB</i>^<i>Tg</i> mice.

<p>(<b>A-D</b>) Representative images of IBA1⁺ cells localized to Thio S-labeled plaques in the superior cortex from B6.<i>APB</i>^<i>Tg</i> (A, B) and D2.<i>APB</i>^<i>Tg</i> (C, D) mice. (<b>E</b>) No significant differences were observed in the number of IBA1⁺ cells surrounding plaques in D2.<i>APB</i>^<i>Tg</i> compared to B6.<i>APB</i>^<i>Tg</i> mice (p = 0.65). (<b>F-G</b>) There was also no obvious difference in the level of astrocyte reactivity (judged by levels of GFAP staining) in regions of plaques in D2.<i>APB</i>^<i>Tg</i> compared to B6.<i>APB</i>^<i>Tg</i> mice. NS = Not significant. Scale bars: A, C = 100μm; B, D, F, G = 20μm.</p

C5 sufficiency does not affect disease state in D2.<i>APB</i>^<i>Tg</i> mice.

<p>(<b>A-C</b>) Plaque deposition is unchanged in D2.<i>APB</i>^<i>Tg</i>.<i>C5</i>^<i>B6</i> (<i>C5</i>^<i>B6</i>) mice compared to D2.<i>APB</i>^<i>Tg</i> (C5^D2) mice (p = 0.251). Similarly to D2.<i>APB</i>^<i>Tg</i> mice, D2.<i>APB</i>^<i>Tg</i>.<i>C5</i>^<i>B6</i> show significantly less plaque deposition compared to B6.<i>APB</i>^<i>Tg</i> (B6) mice (p = 0.001). (<b>D-F</b>) The number of IBA1⁺ cells surrounding plaques in not different in D2.<i>APB</i>^<i>Tg</i>.<i>C5</i>^<i>B6</i> (<i>C5</i>^<i>B6</i>) mice compared to either D2.<i>APB</i>^<i>Tg</i> (C5^D2) or B6.<i>APB</i>^<i>Tg</i> (B6) mice (p = 0.354 and p = 0.087 respectively). (<b>G-I</b>) No significant difference was observed in NeuN⁺ cells in the cortex in D2.<i>APB</i>^<i>Tg</i>.<i>C5</i>^<i>B6</i> (<i>C5</i>^<i>B6</i>) mice compared to D2.<i>APB</i>^<i>Tg</i> (C5^D2, p = 0.44). Scale bars: A, B = 100μm; D, E = 20μm; G, H = 50μm.</p

No overt changes in cortical neurons in D2.<i>APB</i>^<i>Tg</i> mice.

<p>(<b>A-D</b>) NeuN+ cells were counted in B6 (A), B6.<i>APB</i>^<i>Tg</i> (B), D2 (C) and D2.<i>APB</i>^<i>Tg</i> (D) mice. Representative images are shown and all mice showed normal neuronal morphology in three different regions of the cortex. (E) No significant difference was observed in pTau aggregates (AT8 antibody, red) in D2.<i>APB</i>^<i>Tg</i> mice (E) compared to B6.<i>APB</i>^<i>Tg</i> mice. (F) NeuN+ cells were counted in each of the 4 cohorts, in 3 discrete cortical areas (4 mice per cohort). No statistically significant difference is seen between the different cohorts (p = 0.053). Scale bars: A-D = 50μm, E = 10μm.</p

Plaque deposition is dramatically reduced in D2.<i>APB</i>^<i>Tg</i> mice compared to B6.APB^<i>Tg</i> mice.

<p>(<b>A-D</b>) Representative images of ThioS-labeled plaques in the superior cortex from B6.<i>APB</i>^<i>Tg</i> (A, B) and D2.<i>APB</i>^<i>Tg</i> (C, D) mice. Boxed regions in A and C are enlarged in B and D respectively. (<b>F</b>) Plaque counts from the cortex confirm a significant reduction in plaque number in D2.<i>APB</i>^<i>Tg</i> compared to B6.<i>APB</i>^<i>Tg</i> mice (p = 0.001). (<b>E</b>) Aβ42 levels (by ELISA) are significantly reduced in D2.<i>APB</i>^<i>Tg</i> compared to B6.<i>APB</i>^<i>Tg</i> mice (p = 0.0086). Scale bars: A, B = 200μm; C-D = 50μm.</p

Antileukemic activity of valproic acid in chronic lymphocytic leukemia B cells defined by microarray analysis.

Epigenetic code modifications by histone deacetylase inhibitors have recently been proposed as potential new therapies for hematological malignancies. Chronic lymphocytic leukemia (CLL) remains incurable despite the introduction of new treatments. CLL B cells are characterized by an apoptosis defect rather than excessive proliferation, but proliferation centers have been found in organs such as the bone marrow and lymph nodes. In this study, we analyzed gene expression modifications in CLL B cells after treatment with valproic acid (VPA), a well-tolerated anti-epileptic drug with HDAC inhibitory activity. CLL B cells obtained from 14 patients were treated in vitro with a concentration of 1 mM VPA for 4 h. VPA effects on gene expression were thereafter studied using Affymetrix technology, and some identified genes were validated by real-time PCR and western blot. We observed that VPA induced apoptosis by downregulating several anti-apoptotic genes and by upregulating pro-apoptotic genes. Furthermore, VPA significantly increased chemosensitivity to fludarabine, flavopiridol, bortezomib, thalidomide and lenalidomide. VPA inhibited the proliferation of CpG/IL2-stimulated CLL B cells and modulated many cell cycle messenger RNAs. In conclusion, exposure of CLL B cells to VPA induced apoptosis, potentiated chemotherapeutic agent effects and inhibited proliferation. These data strongly suggest the use of VPA in CLL treatment, particularly in combination with antileukemia agents.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe