Pathological histone acetylation in Parkinson's disease: Neuroprotection and inhibition of microglial activation through SIRT 2 inhibition

Parkinson's disease (PD) is associated with degeneration of nigrostriatal neurons due to intracytoplasmic inclusions composed predominantly of a synaptic protein called α-synuclein. Accumulations of α-synuclein are thought to 'mask' acetylation sites on histone proteins, inhibiting the action of histone acetyltransferase (HAT) enzymes in their equilibrium with histone deacetylases (HDACs), thus deregulating the dynamic control of gene transcription. It is therefore hypothesised that the misbalance in the actions of HATs/HDACs in neurodegeneration can be rectified with the use of HDAC inhibitors, limiting the deregulation of transcription and aiding neuronal homeostasis and neuroprotection in disorders such as PD. Here we quantify histone acetylation in the Substantia Nigra pars compacta (SNpc) in the brains of control, early and late stage PD cases to determine if histone acetylation is a function of disease progression. PD development is associated with Braak-dependent increases in histone acetylation. Concurrently, we show that as expected disease progression is associated with reduced markers of dopaminergic neurons and increased markers of activated microglia. We go on to demonstrate that in vitro, degenerating dopaminergic neurons exhibit histone hypoacetylation whereas activated microglia exhibit histone hyperacetylation. This suggests that the disease-dependent increase in histone acetylation observed in human PD cases is likely a combination of the contributions of both degenerating dopaminergic neurons and infiltrating activated microglia. The HDAC SIRT 2 has become increasingly implicated as a novel target for mediation of neuroprotection in PD: the neuronal and microglial specific effects of its inhibition however remain unclear. We demonstrate that SIRT 2 expression in the SNpc of PD brains remains relatively unchanged from controls and that SIRT 2 inhibition, via AGK2 treatment of neuronal and microglial cultures, results in neuroprotection of dopaminergic neurons and reduced activation of microglial cells. Taken together, here we demonstrate that histone acetylation is disease-dependently altered in PD, likely due the effects of dopaminergic neurodegeneration and microglial infiltration; yet SIRT 2 remains relatively unaltered with disease. Given the stable nature of SIRT 2 expression with disease and the effects of SIRT 2 inhibitor treatment on degenerating dopaminergic neurons and activated microglia detected in vitro, SIRT 2 inhibitors warrant further investigation as potential therapeutics for the treatment of the PD

The Histone Deacetylase Inhibitor Nicotinamide Exacerbates Neurodegeneration in the Lactacystin Rat Model of Parkinson's Disease

Histone hypoacetylation is associated with dopaminergic neurodegeneration in Parkinson's disease (PD), due to an imbalance in the activities of the enzymes responsible for histone (de)acetylation. Correction of this imbalance, with histone deacetylase (HDAC) inhibiting agents, could be neuroprotective. We therefore hypothesise that nicotinamide, being a selective inhibitor of HDAC class III as well as having modulatory effects on mitochondrial energy metabolism, would be neuroprotective in the lactacystin rat model of PD, which recapitulates the formation of neurotoxic accumulation of altered proteins within the substantia nigra to cause progressive dopaminergic cell death. Rats received nicotinamide for 28 days, starting 7 days after unilateral injection of the irreversible proteasome inhibitor, lactacystin, into the substantia nigra. Longitudinal motor behavioural testing and structural MRI were used to track changes in this model of PD, and assessment of nigrostriatal integrity, histone acetylation, and brain gene expression changes post-mortem used to quantify nicotinamide induced neuroprotection. Counterintuitively, nicotinamide dose-dependently exacerbated neurodegeneration of dopaminergic neurons, behavioural deficits, and structural brain changes in the lactacystin-lesioned rat. Nicotinamide treatment induced histone hyperacetylation and overexpression of numerous neurotrophic and anti-apoptotic factors in the brain, yet failed to result in neuroprotection, rather exacerbated dopaminergic pathology. These findings highlight the importance of inhibitor specificity within HDAC isoforms for therapeutic efficacy in PD, demonstrating the contrasting effects of HDAC class III inhibition upon cell survival in this animal model of the disease. This article is protected by copyright. All rights reserved

Mean expression of the X-chromosome is associated with neuronal density

peer reviewedBackground: Neurodegenerative diseases are characterized by key features such as loss of neurons, astrocytosis, and microglial activation/proliferation. These changes cause differences in the density of cell types between control and disease subjects, confounding results from gene expression studies. Chromosome X (ChrX) is known to be specifically important in the brain. We hypothesized the existence of a chromosomal signature of gene expression associated with the X-chromosome for neurological conditions not normally associated with that chromosome. The hypothesis was investigated using publicly available microarray datasets from studies on Parkinson's disease, Alzheimer's disease, and Huntington's disease. Data were analyzed using Chromowave, an analytical tool for detecting spatially extended expression changes along chromosomes. To examine associations with neuronal density and astrocytosis, the expression of cell specific reporter genes was extracted. The association between these genes and the expression patterns extracted by Chromowave was then analyzed. Further analyses of the X:Autosome ratios for laser dissected neurons, microglia cultures and whole tissue were performed to detect cell specific differences. Results: We observed an extended pattern of low expression of ChrX consistent in all the neurodegenerative disease brain datasets. There was a strong correlation between mean ChrX expression and the pattern extracted from the autosomal genes representing neurons, but not with mean autosomal expression. No chromosomal patterns associated with the neuron specific genes were found on other chromosomes. The chromosomal expression pattern was not present in datasets from blood cells. The X:Autosome expression ratio was also higher in neuronal cells than in tissues with a mix of cell types. Conclusions: The results suggest that neurological disorders show as a reduction in mean expression of many genes along ChrX. The most likely explanation for this finding relates to the documented general up-regulation of ChrX in brain tissue which, this work suggests, occurs primarily in neurons. If validated, this cell specific ChrX expression warrants further research as understanding the biological reasons and mechanisms for this expression, may help to elucidate a connection with the development of neurodegenerative disorders

L-DOPA functionalized, multi-branched gold nanoparticles as brain-targeted nano-vehicles

The blood–brain barrier (BBB) is a protective endothelial barrier lining the brain microvasculature which prevents brain delivery of therapies against brain diseases. Hence, there is an urgent need to develop vehicles which efficiently penetrate the BBB to deliver therapies into the brain. The drug L-DOPA efficiently and specifically crosses the BBB via the large neutral amino acid transporter (LAT)-1 protein to enter the brain. Thus, we synthesized L-DOPA-functionalized multi-branched nanoflower-like gold nanoparticles (L-DOPA-AuNFs) using a seed-mediated method involving catechols as a direct reducing-cum-capping agent, and examined their ability to cross the BBB to act as brain-penetrating nanovehicles. We show that L-DOPA-AuNFs efficiently penetrate the BBB compared to similarly sized and shaped AuNFs functionalized with a non-targeting ligand. Furthermore, we show that L-DOPA-AuNFs are efficiently internalized by brain macrophages without inducing inflammation. These results demonstrate the application of L-DOPA-AuNFs as a non-inflammatory BBB-penetrating nanovehicle to efficiently deliver therapies into the brain

Multibranched Gold Nanoparticles with Intrinsic LAT-1 Targeting Capabilities for Selective Photothermal Therapy of Breast Cancer

Because of the critical role of the large neutral amino acid transporter-1 (LAT-1) in promoting tumor growth and proliferation, it is fast emerging as a highly attractive biomarker for the imaging and treatment of human malignancies, including breast cancer. While multibranched gold nanoparticles (AuNPs) have emerged as a promising modality in the photothermal therapy (PTT) of cancers, some of the key challenges limiting their clinical translation lie in the need to develop reproducible and cost-effective synthetic methods as well as the selective accumulation of sufficient AuNPs at tumor sites. In this study, we report a simple and direct seed-mediated synthesis of monodispersed multibranched AuNPs using the catechol-containing LAT-1 ligands, L- and D-dopa, to confer active cancer targeting. This route obviates the need for additional conjugation with targeting moieties such as peptides or antibodies. Nanoflower-like AuNPs (AuNF) with diameters of approximately 46, 70, and 90 nm were obtained and were found to possess excellent colloidal stability and biocompatibility. A significantly higher intracellular accumulation of the L- and D-dopa functionalized AuNFs was observed in a panel of breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468, and MDA-MB-453) when compared to the nontargeting control AuNFs synthesized with dopamine and 4-ethylcatechol. Importantly, no significant difference in uptake between the targeting and nontargeting AuNFs was observed in a non-tumorigenic MCF-10A breast epithelial cell line, hence demonstrating tumor selectivity. For PTT of breast cancer, Ag+ was introduced during synthesis to obtain L-dopa functionalized nanourchin-like AuNPs (AuNUs) with strong near-infrared (NIR) absorbance. The L-dopa functionalized AuNUs mediated selective photothermal ablation of the triple negative MDA-MB-231 breast cancer cell line and sensitized the cells to the anticancer drugs cisplatin and docetaxel. This work brings forward an effective strategy for the facile preparation of cancer targeting multibranched AuNPs with potential for the in vivo PTT of breast cancer

Correlations of Behavioral Deficits with Brain Pathology Assessed through Longitudinal MRI and Histopathology in the R6/2 Mouse Model of HD

Huntington's disease (HD) is caused by the expansion of a CAG repeat in the huntingtin (HTT) gene. The R6/2 mouse model of HD expresses a mutant version of exon 1 HTT and develops motor and cognitive impairments, a widespread huntingtin (HTT) aggregate pathology and brain atrophy. Despite the vast number of studies that have been performed on this model, the association between the molecular and cellular neuropathology with brain atrophy, and with the development of behavioral phenotypes remains poorly understood. In an attempt to link these factors, we have performed longitudinal assessments of behavior (rotarod, open field, passive avoidance) and of regional brain abnormalities determined through magnetic resonance imaging (MRI) (whole brain, striatum, cortex, hippocampus, corpus callosum), as well as an end-stage histological assessment. Detailed correlative analyses of these three measures were then performed. We found a gender-dependent emergence of motor impairments that was associated with an age-related loss of regional brain volumes. MRI measurements further indicated that there was no striatal atrophy, but rather a lack of striatal growth beyond 8 weeks of age. T2 relaxivity further indicated tissue-level changes within brain regions. Despite these dramatic motor and neuroanatomical abnormalities, R6/2 mice did not exhibit neuronal loss in the striatum or motor cortex, although there was a significant increase in neuronal density due to tissue atrophy. The deposition of the mutant HTT (mHTT) protein, the hallmark of HD molecular pathology, was widely distributed throughout the brain. End-stage histopathological assessments were not found to be as robustly correlated with the longitudinal measures of brain atrophy or motor impairments. In conclusion, modeling pre-manifest and early progression of the disease in more slowly progressing animal models will be key to establishing which changes are causally related. © 2013 Rattray et al

Label-free TOF-SIMS imaging of sulfur producing enzymes inside microglia cells following exposure to silver nanowires

There are no methods sensitive enough to detect enzymes within cells, without the use of analyte labelling. Here we show that it is possible to detect protein ion signals of three different H2S-synthesizing enzymes inside microglia after pre-treatment with silver nanowires (AgNW) using time of flight-secondary ion mass spectrometry (TOF-SIMS). Protein fragment ions, including the fragment of amino acid (C4H8N+ - 70 amu), fragments of the sulfur producing cystathionine-containing enzymes and the Ag+ ion signal could be detected without the use of any labels; the cells were mapped using the C4H8N+ amino acid fragment. Scanning electron microscopy imaging and energy dispersive x-ray chemical analysis showed that the AgNWs were inside the same cells imaged by TOF-SIMS and transformed chemically into crystalline Ag2S within cells in which the sulfur producing proteins were detected. The presence of these sulfur producing cystathionine-containing enzymes within the cells was confirmed by Western Blots and confocal microscopy images of fluorescently labelled antibodies against the sulfur producing enzymes. Label-free ToF-SIMS is very promising for the label-free identification of H2S-contributing enzymes and their cellular localization in biological systems. The technique could in future be used to identify which of these enzymes are most contributory

Osteoarthritis: quality of life, comorbidities, medication and health service utilization assessed in a large sample of primary care patients

<p>Abstract</p> <p>Objective</p> <p>To assess the gender related impact of osteoarthritis (OA) on quality of life (QoL) and health service utilization (HSU) of primary care patients in Germany.</p> <p>Methods</p> <p>Cross sectional study with 1250 OA patients attending 75 primary care practices from March to May 2005. QoL was assessed using the GERMAN-AIMS2-SF. Data about comorbidities, prescriptions, health service utilization, and physical activity were obtained by questioning patients or from the patients' medical files. Depression was assessed by means of the Patient Health Questionnaire (PHQ-9).</p> <p>Results</p> <p>1021 (81.7%) questionnaires were returned. 347 (34%) patients were male. Impact of OA on QoL was different between gender: women achieved significantly higher scores in the AIMS 2-SF dimensions lower body (p < 0.01), symptom (p < 0.01), affect (p < 0.01) and work (p < 0.05). Main predictors of pain and disability were a high score in the "upper body "scale of the AIMS2-SF (beta = 0.280; p < 0.001), a high score in the PHQ-9 (beta = 0.214; p < 0.001), duration of OA (beta = 0.097; p = 0.004), age (beta = 0.090; p = 0.023) and the BMI (beta = 0.069; p = 0.034). Predictors of pain and disability did not differ between gender. 18.8 % of men and 19.7% of women had a concomitant depression. However, no gender differences occurred. Women visited their GP (mean 5.61 contacts in 6 months) more often than men (mean 4.08; p < 0.01); visits to orthopedics did not differ between gender.</p> <p>Conclusion</p> <p>The extent to which OA impacts men and women differs in primary care patients. This might have resulted in the revealed differences in the pharmacological treatment and the HSU. Further research is needed to confirm our findings and to assess causality.</p

The attitudes, beliefs and behaviours of GPs regarding exercise for chronic knee pain: a systematic review

<p>Abstract</p> <p>Background</p> <p>Joint pain, specifically chronic knee pain (CKP), is a frequent cause of chronic pain and limitation of function and mobility among older adults. Multiple evidence-based guidelines recommend exercise as a first-line treatment for all patients with CKP or knee osteoarthritis (KOA), yet healthcare practitioners' attitudes and beliefs may limit their implementation. This systematic review aims to identify the attitudes, beliefs and behaviours of General Practitioners (GPs) regarding the use of exercise for CKP/KOA.</p> <p>Methods</p> <p>We searched four electronic databases between inception and January 2008, using subject headings to identify studies examining the attitudes, beliefs or behaviours of GPs regarding the use of exercise for the treatment of CKP/KOA in adults aged over 45 years in primary care. Studies referring to patellofemoral pain syndrome or CKP secondary to other causes or that occurring in a prosthetic joint were excluded. Once inclusion and exclusion criteria were applied, study data were extracted and summarised. Study quality was independently reviewed using two assessment tools.</p> <p>Results</p> <p>From 2135 potentially relevant articles, 20 were suitable for inclusion. A variety of study methodologies and approaches to measuring attitudes beliefs and behaviours were used among the studies. Quality assessment revealed good reporting of study objective, type, outcome factors and, generally, the sampling frame. However, criticisms included use of small sample sizes, low response rates and under-reporting of non-responder factors. Although 99% of GPs agreed that exercise should be used for CKP/KOA and reported ever providing advice or referring to a physiotherapist, up to 29% believed that rest was the optimum management approach. The frequency of actual provision of exercise advice or physiotherapy referral was lower. Estimates of provision of exercise advice and physiotherapy referral were generally higher for vignette-based studies (exercise advice 9%-89%; physiotherapy referral 44%-77%) than reviews of actual practice (exercise advice 5%-52%; physiotherapy referral 13-63%). <it>A</it><it>dvice to exercise </it>and exercise <it>prescription </it>were not clearly differentiated.</p> <p>Conclusions</p> <p>Attitudes and beliefs of GPs towards exercise for CKP/KOA vary widely and exercise appears to be underused in the management of CKP/KOA. Limitations of the evidence base include the paucity of studies directly examining attitudes of GPs, poor methodological quality, limited generalisability of results and ambiguity concerning GPs' expected roles. Further investigation is required of the roles of GPs in using exercise as first-line management of CKP/KOA.</p