Plasma proteins predict conversion to dementia from prodromal disease.

BACKGROUND: The study aimed to validate previously discovered plasma biomarkers associated with AD, using a design based on imaging measures as surrogate for disease severity and assess their prognostic value in predicting conversion to dementia. METHODS: Three multicenter cohorts of cognitively healthy elderly, mild cognitive impairment (MCI), and AD participants with standardized clinical assessments and structural neuroimaging measures were used. Twenty-six candidate proteins were quantified in 1148 subjects using multiplex (xMAP) assays. RESULTS: Sixteen proteins correlated with disease severity and cognitive decline. Strongest associations were in the MCI group with a panel of 10 proteins predicting progression to AD (accuracy 87%, sensitivity 85%, and specificity 88%). CONCLUSIONS: We have identified 10 plasma proteins strongly associated with disease severity and disease progression. Such markers may be useful for patient selection for clinical trials and assessment of patients with predisease subjective memory complaints.

MuRF-1 and atrogin-1 protein expression and quadriceps fiber size and muscle mass in stable patients with COPD.

INTRODUCTION: Animal studies demonstrate the importance of the E3 ubiquitin ligases, Muscle RING-Finger Protein 1 (MuRF-1) and atrogin-1, in muscle protein degradation during acute muscle atrophy. Small clinical studies suggest MuRF-1 and atrogin-1 expression in the quadriceps muscle is also increased in stable patients with Chronic Obstructive Pulmonary Disease compared to controls. However, it remains unclear whether these ligases have a role in maintaining a muscle-wasted state in COPD patients. METHODS: 32 stable COPD patients (16 with a low fat-free mass index (FFMI), 16 with a normal FFMI) and 15 controls underwent lung function and quadriceps strength tests and a percutaneous quadriceps biopsy. Quadriceps MuRF-1 and atrogin-1 protein were quantified with western blotting. Quadriceps fiber cross-sectional area (CSA) and fiber proportions were determined by immunohistochemistry on muscle sections. MuRF-1 and atrogin-1 levels were compared between COPD patients with and without a low FFMI, and between patients and controls, and correlations between MuRF-1 and atrogin-1 levels and quadriceps fiber CSA in the patients were investigated. RESULTS: Atrogin-1 protein levels were lower in patients than controls, but similar in patients with a low and normal FFMI. MuRF-1 levels did not differ between any groups. MuRF-1 and atrogin-1 levels were not associated with quadriceps fiber CSA or quadriceps strength in patients. CONCLUSIONS: Chronic upregulation of ubiquitin ligases was not evident in the quadriceps muscle of stable COPD patients with a low muscle mass. This does not exclude the possibility of transient increases in ubiquitin ligases during acute catabolic episodes.

Downregulation of the serum response factor/miR-1 axis in the quadriceps of patients with COPD.

RATIONALE: Muscle atrophy confers a poor prognosis in patients with chronic obstructive pulmonary disease (COPD), yet the molecular pathways responsible are poorly characterised. Muscle-specific microRNAs and serum response factor (SRF) are important regulators of muscle phenotype that contribute to a feedback system to regulate muscle gene expression. The role of these factors in the skeletal muscle dysfunction that accompanies COPD is unknown. METHODS: 31 patients with COPD and 14 healthy age-matched controls underwent lung and quadriceps function assessments, measurement of daily activity and a percutaneous quadriceps muscle biopsy. The expression of muscle-specific microRNAs, myosin heavy chains and components of the serum response factor signalling pathway were determined by qPCR. RESULTS: A reduction in expression of miR-1 (2.5-fold, p=0.01) and the myocardin-related transcription factors (MRTFs) A and B was observed in patients compared with controls (MRTF-A mRNA: twofold, p=0.028; MRTF-B mRNA: fourfold, p=0.011). miR-1 expression was associated with smoking history, lung function, fat-free mass index, 6 min walk distance and percentage of type 1 fibres. miR-133 and miR-206 were negatively correlated with daily physical activity. Insulin-like growth factor 1 mRNA was increased in the patients and miR-1 was negatively correlated with phosphorylation of the kinase Akt. Furthermore, the protein levels of histone deacetylase 4, another miR-1 target, were increased in the patients. CONCLUSIONS: Downregulation of the activity of the MRTF-SRF axis and the expression of muscle-specific microRNAs, particularly miR-1, may contribute to COPD-associated skeletal muscle dysfunction.

Alzheimer's disease biomarker discovery using in silico literature mining and clinical validation.

BACKGROUND: Alzheimer's Disease (AD) is the most widespread form of dementia in the elderly but despite progress made in recent years towards a mechanistic understanding, there is still an urgent need for disease modification therapy and for early diagnostic tests. Substantial international efforts are being made to discover and validate biomarkers for AD using candidate analytes and various data-driven 'omics' approaches. Cerebrospinal fluid is in many ways the tissue of choice for biomarkers of brain disease but is limited by patient and clinician acceptability, and increasing attention is being paid to the search for blood-based biomarkers. The aim of this study was to use a novel in silico approach to discover a set of candidate biomarkers for AD. METHODS: We used an in silico literature mining approach to identify potential biomarkers by creating a summarized set of assertional metadata derived from relevant legacy information. We then assessed the validity of this approach using direct assays of the identified biomarkers in plasma by immunodetection methods. RESULTS: Using this in silico approach, we identified 25 biomarker candidates, at least three of which have subsequently been reported to be altered in blood or CSF from AD patients. Two further candidate biomarkers, indicated from the in silico approach, were choline acetyltransferase and urokinase-type plasminogen activator receptor. Using immunodetection, we showed that, in a large sample set, these markers are either altered in disease or correlate with MRI markers of atrophy. CONCLUSIONS: These data support as a proof of concept the use of data mining and in silico analyses to derive valid biomarker candidates for AD and, by extension, for other disorders.

Mild stress of caffeine increased mtDNA content in skeletal muscle cells: the interplay between Ca2+ transients and nitric oxide.

Caffeine increases mitochondrial biogenesis in myotubes by evoking Ca(2+) transients. Nitric oxide (NO) also induces mitochondrial biogenesis in skeletal muscle cells via upregulation of AMP-activated protein kinase (AMPK) activity and PGC-1α. However, the interplay and timing sequence between Ca(2+) transients and NO releases remain unclear. Herein, we tested the hypothesis that caffeine-evoked Ca(2+) transients triggered NO production to increase mtDNA in skeletal muscle cells. Ca(2+) transients were recorded with Fura-2 AM and confocal microscopy; mtDNA staining, mitochondrial membrane potential and NO level were determined using fluorescent probes PicoGreen, tetramethylrhodamine methyl ester (TMRM) and DAF-FM, respectively. In primary cultured myotubes, a subtle and moderate stress of caffeine increased mtDNA exclusively. Mitochondrial membrane potential and mtDNA were increased by 1 mM as well as 5 mM caffeine, whereas 10 mM caffeine did not change the fluorescence intensity of PicoGreen and TMRM. NO level in myocytes increased gradually following the first jump of Ca(2+) transients evoked by caffeine (5 mM) till the end of recording, when Fura-2 indicated that Ca(2+) transients recovered partly and even disappeared. Importantly, nitric oxide synthase (NOS) inhibitor (L-NAME) suppressed caffeine-induced mtDNA biogenesis, whereas NO donor (DETA-NO) increased mtDNA content. These data strongly suggest that caffeine-induced mtDNA biogenesis is dose-sensitive and dependent on a certain level of stress. Further, an increasing level of NO following Ca(2+) transients is required for caffeine-induced mtDNA biogenesis. Additionally, Ca(2+) transients, a usual and first response to caffeine, was either suppressed or attenuated by L-NAME, DETA-NO, AICAR and U0126, suggesting an inability to control [Ca(2+)](i) in these treated cells. There may be an important interplay between NO and Ca(2+) transients in intracellular signaling system involving NOS, AMPK and MEK.

p38 mitogen-activated protein kinase is not activated in the quadriceps of patients with stable chronic obstructive pulmonary disease.

Quadriceps weakness is associated with a poor prognosis in COPD. Several data suggest that immobility and muscle wasting may be related through up-regulation of the p38 Mitogen associated protein kinase (MAPK) signalling pathway. We therefore hypothesised that this might occur in the quadriceps of COPD patients. We studied 105 stable COPD outpatients (mean FEV(1) 43.9% predicted) and 27 age and gender matched controls. We measured fat-free mass, quadriceps strength and daily physical activity using triaxial accelerometry. A quadriceps biopsy was obtained in which components of the p38-MAPK signalling pathway were examined. Patients had reduced fat-free mass index (16.1 (2.2) kg/m(2) vs 17.2 (2.2) kg/m(2), p = 0.02) and exhibited quadriceps weakness (mean (SD) maximal voluntary contraction force 72.1% (18.7) predicted and 82.3% (7.1) predicted for patients and controls respectively, p = 0.01). Physical activity was significantly reduced in the patient group; in particular mean (SD) locomotion time was 101 (48) minutes/12 hours in controls and 48 (31) minutes in patients (p < 0.0001). However, in biopsies obtained from these patients, no differences were observed for total or phosphorylated HSP27 or p38 MAPK protein or p38 MAPK mRNA (MAPK14); of the downstream products both GADD45ß and c-jun mRNA were reduced in COPD patients while c-myc was not different from controls. No parameter correlated with physiological data within the patient group. We conclude that, despite the presence of reduced fat-free mass, quadriceps weakness and inactivity, p38 MAPK signalling was not up-regulated in skeletal muscle of stable out-patients with COPD.

Upregulation of PKD1L2 provokes a complex neuromuscular disease in the mouse.

Following a screen for neuromuscular mouse mutants, we identified ostes, a novel N-ethyl N-nitrosourea-induced mouse mutant with muscle atrophy. Genetic and biochemical evidence shows that upregulation of the novel, uncharacterized transient receptor potential polycystic (TRPP) channel PKD1L2 (polycystic kidney disease gene 1-like 2) underlies this disease. Ostes mice suffer from chronic neuromuscular impairments including neuromuscular junction degeneration, polyneuronal innervation and myopathy. Ectopic expression of PKD1L2 in transgenic mice reproduced the ostes myopathic changes and, indeed, caused severe muscle atrophy in Tg(Pkd1l2)/Tg(Pkd1l2) mice. Moreover, double-heterozygous mice (ostes/+, Tg(Pkd1l2)/0) suffer from myopathic changes more profound than each heterozygote, indicating positive correlation between PKD1L2 levels and disease severity. We show that, in vivo, PKD1L2 primarily associates with endogenous fatty acid synthase in normal skeletal muscle, and these proteins co-localize to costameric regions of the muscle fibre. In diseased ostes/ostes muscle, both proteins are upregulated, and ostes/ostes mice show signs of abnormal lipid metabolism. This work shows the first role for a TRPP channel in neuromuscular integrity and disease.

Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition in which motoneurons of the spinal cord and motor cortex die, resulting in progressive paralysis. This condition has no cure and results in eventual death, usually within 1-5 years of diagnosis. Although the specific etiology of ALS is unknown, 20% of familial cases of the disease carry mutations in the gene encoding Cu/Zn superoxide dismutase-1 (SOD1). Transgenic mice overexpressing human mutant SOD1 have a phenotype and pathology that are very similar to that seen in human ALS patients. Here we show that treatment with arimoclomol, a coinducer of heat shock proteins (HSPs), significantly delays disease progression in mice expressing a SOD1 mutant in which glycine is substituted with alanine at position 93 (SOD1(G93A)). Arimoclomol-treated SOD1(G93A) mice show marked improvement in hind limb muscle function and motoneuron survival in the later stages of the disease, resulting in a 22% increase in lifespan. Pharmacological activation of the heat shock response may therefore be a successful therapeutic approach to treating ALS, and possibly other neurodegenerative diseases.

Mitochondrial genes are altered in blood early in Alzheimer's disease.

Although mitochondrial dysfunction is a consistent feature of Alzheimer's disease in the brain and blood, the molecular mechanisms behind these phenomena are unknown. Here we have replicated our previous findings demonstrating reduced expression of nuclear-encoded oxidative phosphorylation (OXPHOS) subunits and subunits required for the translation of mitochondrial-encoded OXPHOS genes in blood from people with Alzheimer's disease and mild cognitive impairment. Interestingly this was accompanied by increased expression of some mitochondrial-encoded OXPHOS genes, namely those residing closest to the transcription start site of the polycistronic heavy chain mitochondrial transcript (MT-ND1, MT-ND2, MT-ATP6, MT-CO1, MT-CO2, MT-C03) and MT-ND6 transcribed from the light chain. Further we show that mitochondrial DNA copy number was unchanged suggesting no change in steady-state numbers of mitochondria. We suggest that an imbalance in nuclear and mitochondrial genome-encoded OXPHOS transcripts may drive a negative feedback loop reducing mitochondrial translation and compromising OXPHOS efficiency, which is likely to generate damaging reactive oxygen species.

Plasma based markers of [11C] PiB-PET brain amyloid burden.

Changes in brain amyloid burden have been shown to relate to Alzheimer's disease pathology, and are believed to precede the development of cognitive decline. There is thus a need for inexpensive and non-invasive screening methods that are able to accurately estimate brain amyloid burden as a marker of Alzheimer's disease. One potential method would involve using demographic information and measurements on plasma samples to establish biomarkers of brain amyloid burden; in this study data from the Alzheimer's Disease Neuroimaging Initiative was used to explore this possibility. Sixteen of the analytes on the Rules Based Medicine Human Discovery Multi-Analyte Profile 1.0 panel were found to associate with [(11)C]-PiB PET measurements. Some of these markers of brain amyloid burden were also found to associate with other AD related phenotypes. Thirteen of these markers of brain amyloid burden--c-peptide, fibrinogen, alpha-1-antitrypsin, pancreatic polypeptide, complement C3, vitronectin, cortisol, AXL receptor kinase, interleukin-3, interleukin-13, matrix metalloproteinase-9 total, apolipoprotein E and immunoglobulin E--were used along with co-variates in multiple linear regression, and were shown by cross-validation to explain >30% of the variance of brain amyloid burden. When a threshold was used to classify subjects as PiB positive, the regression model was found to predict actual PiB positive individuals with a sensitivity of 0.918 and a specificity of 0.545. The number of APOE [Symbol: see text] 4 alleles and plasma apolipoprotein E level were found to contribute most to this model, and the relationship between these variables and brain amyloid burden was explored.

Yin Yang 1 expression and localisation in quadriceps muscle in COPD.

INTRODUCTION: Yin Yang 1 (YY1) is a transcriptional repressor that inhibits muscle gene expression and myogenesis. YY1 has not previously been investigated in the skeletal muscle of patients with COPD. The aims of this study were to investigate YY1 expression and localisation in the quadriceps muscle of COPD patients compared to healthy age-matched controls, and examine the relationship between YY1 expression and localisation and quadriceps muscle fibre cross-sectional area (CSA) in COPD patients. PATIENTS AND METHODS: 15 COPD patients and 8 age-matched controls underwent lung and quadriceps function assessments and a percutaneous quadriceps biopsy. Quadriceps muscle fibre CSA and fibre proportions and YY1 localisation were determined by immunofluorescence. YY1 was immunoprecipitated from muscle and YY1 levels assessed by western blotting. RESULTS: YY1 levels were inversely correlated with type IIx and type I fibre CSA in patients and controls, though YY1 levels were not significantly different between the groups. Nuclear localisation of YY1 was demonstrated in the patients but not in controls. CONCLUSION: YY1 expression is associated with smaller quadriceps fibre CSA in COPD and nuclear localisation of YY1 was found in muscle of patients but not controls. Regulation of YY1 appears altered in COPD and may be implicated in COPD-related muscle atrophy.

Proteome-based plasma markers of brain amyloid-ß deposition in non-demented older individuals.

Blood-based markers reflecting core pathological features of Alzheimer's disease (AD) in pre-symptomatic individuals are likely to accelerate the development of disease-modifying treatments. Our aim was to discover plasma proteins associated with brain amyloid-ß (Aß) burden in non-demented older individuals. We performed discovery-phase experiments using two dimensional gel electrophoresis (2DGE) and mass spectrometry-based proteomic analysis of plasma in combination with 11C-PiB PET imaging of the brain in samples collected 10 years prior to the PET scans. Confirmatory studies used ELISA assays in a separate set of blood samples obtained within a year of the PET scans. We observed that a panel of 18 2DGE plasma protein spots effectively discriminated between individuals with high and low brain Aß. Mass spectrometry identified these proteins, many of which have established roles in Aß clearance, including a strong signal from apolipoprotein-E (ApoE). In validation-phase studies, we observed a strong association between plasma ApoE concentration and Aß burden in the medial temporal lobe. Targeted voxel-based analysis localized this association to the hippocampus and entorhinal cortex. APOE ε4 carriers also showed greater Aß levels in several brain regions relative to ε4 non-carriers. These results suggest that both peripheral concentration of ApoE protein and APOE genotype are related to early neuropathological changes in brain regions vulnerable to AD pathology even in the non-demented elderly. Our strategy combining proteomics with in vivo brain amyloid imaging holds promise for the discovery of biologically relevant peripheral markers in those at risk for AD.

Mechano-growth factor, an IGF-I splice variant, rescues motoneurons and improves muscle function in SOD1(G93A) mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneuron degeneration. Although viral delivery of IGF-I has shown therapeutic efficacy in the SOD1(G93A) mouse model of ALS, clinical trials of IGF-I in ALS patients have led to conflicting results. Here we examine the effects of an IGF-I splice variant, mechano-growth factor (MGF) which has previously been shown to have greater neuroprotective effects than IGF-I in a number of models of neurodegeneration. A mammalian expression plasmid containing either MGF or, for comparison, the IGF-I cDNA sequence was delivered to the hindlimb muscles of SOD1(G93A) mice at 70 days of age, at symptom onset. Treatment with either IGF-I or MGF resulted in a significant improvement in hindlimb muscle strength, and an increase in motor unit and motoneuron survival. Significantly more motoneurons survived in MGF treated mice.

Expresión y localización del factor de transcripción Yin Yang 1 en el músculo cuádriceps en la enfermedad pulmonar obstructiva crónica / Yin Yang 1 expression and localisation in quadriceps muscle in COPD

Introducción: El Ying Yang 1 (YY1) es un factor de transcripción represor que inhibe la expresión génicamuscular y la miogénesis. Este factor no se ha investigado previamente este factor no se ha investigado enel músculo esquelético de pacientes con enfermedad pulmonar obstructiva crónica (EPOC). Los objetivosdel presente estudio fueron investigar la expresión de YY1 y su localización en el músculo cuádricepsde pacientes con EPOC, comparado con individuos control sanos, emparejados por edad, y examinar larelación entre la expresión y localización de YY1 en las áreas transversales (AT) de las fibras muscularesdel cuádriceps en pacientes con EPOC.Pacientes y métodos: Se sometió a 15 pacientes con EPOC y a 8 individuos de control, emparejados poredad, a valoraciones de la función pulmonar y del cuádriceps y a una biopsia percutánea de este músculo.Mediante inmunofluorescencia se determinó el AT de las fibras musculares del cuádriceps las proporcionesde fibras y localización de YY1. YY1 se inmunoprecipitó a partir del músculo y sus niveles se evaluaronmediante inmunotransferencia.Resultados: Los niveles de YY1 se correlacionaron inversamente con el AT de las fibras de tipo IIx y de tipo Ien pacientes e individuos de control, aunque los niveles de YY1 no fueron significativamente diferentesentre ambos grupos. En los pacientes, pero no en los individuos control, se demostró la localizaciónnuclear de YY1.Conclusión: La expresión de YY1 se asocia a un AT más pequeña de las fibras del cuádriceps en pacientescon EPOC, en cuyo músculo también se observa una localización nuclear del factor, a diferencia de losindividuos control. La regulación de YY1 parece alterada en la EPOC y podría estar implicada en la atrofiamuscular relacionada con la enfermedad(AU)

Introduction: Yin Yang 1 (YY1) is a transcriptional repressor that inhibits muscle gene expression andmyogenesis. YY1 has not previously been investigated in the skeletal muscle of patients with COPD.The aims of this study were to investigate YY1 expression and localisation in the quadriceps muscle ofCOPD patients compared to healthy age-matched controls, and examine the relationship between YY1expression and localisation and quadriceps muscle fibre cross-sectional area (CSA) in COPD patients.Patients and methods: 15 COPD patients and 8 age-matched controls underwent lung and quadriceps function assessments and a percutaneous quadriceps biopsy. Quadriceps muscle fibre CSA and fibre proportions and YY1 localisation were determined by immunofluorescence. YY1 was immunoprecipitated from muscle and YY1 levels assessed by western blotting.