Exploring effects of Souvenaid on cerebral glucose metabolism in Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is associated with synapse loss. Souvenaid, containing the specific nutrient combination Fortasyn Connect, was designed to improve synapse formation and function. The NL-ENIGMA study explored the effect of Souvenaid on synapse function in early AD by assessing cerebral glucose metabolism (CMRglc) with 18F-fluorodeoxyglucose ([18F]FDG) positron emission tomography (PET). METHODS: We conducted an exploratory double-blind randomized controlled single-center trial. Fifty patients with mild cognitive impairment or mild dementia with evidence of amyloid pathology (cerebrospinal fluid or PET) were stratified for MMSE (20-24 and 25-30) and randomly 1:1 allocated to 24-week daily administration of 125 mL Souvenaid (n = 25) or placebo (n = 25). Dynamic 60-minute [18F]FDG-PET scans (21 frames) with arterial sampling were acquired at baseline and 24 weeks. CMRglc was estimated by quantitative (Ki) and semiquantitative (standardized uptake value ratio, reference cerebellar gray matter) measurements in five predefined regions of interest and a composite region of interest. Change from baseline in CMRglc was compared between treatment groups by analysis of variance, adjusted for baseline CMRglc and MMSE stratum. Additional exploratory outcome parameters included voxel-based analyses by Statistical Parametric Mapping. RESULTS: No baseline differences between treatment groups were found (placebo/intervention: n = 25/25; age 66 ± 8/65 ± 7 years; female 44%/48%; MMSE 25 ± 3/25 ± 3). [18F]FDG-PET data were available for quantitative (placebo n = 19, intervention n = 18) and semiquantitative (placebo n = 20, intervention n = 22) analyses. At follow-up, no change within treatment groups and no statistically significant difference in change between treatment groups in CMRglc in any regions of interest were found by both quantitative and semiquantitative analyses. No treatment effect was found in the cerebellar gray matter using quantitative measures. The additional Statistical Parametric Mapping analyses did not yield consistent differences between treatment groups. DISCUSSION: In this exploratory trial, we found no robust effect of 24-week intervention with Souvenaid on synapse function measured by [18F]FDG-PET. Possible explanations include short duration of treatment.

Additive Effects of Levodopa and a Neurorestorative Diet in a Mouse Model of Parkinson's Disease.

Though Parkinson's disease (PD) clinical picture is generally dominated by motor impairment, non-motor symptoms, such as cognitive decline and gastrointestinal dysfunctions, may develop before motor symptoms and have major effects on quality of life. L-3,4-di-hydroxy-phenylalanine (Levodopa) is the most commonly used treatment of motor symptoms but has serious side-effects with prolonged use and does not stop the degenerative process. Moreover, gastrointestinal dysfunctions interfere with the absorption of levodopa and modify its effectiveness. Since most patients are on levodopa treatment, there is a need for combinational therapies that allow for an effective reduction of both motor and non-motor symptoms. We have recently shown that a diet containing precursors and cofactors required for membrane phospholipid synthesis, as well as prebiotic fibers, had therapeutic effects in a PD mouse model. We now investigate the effects of combined administration of the same diet together with levodopa in the rotenone model of PD. Mice were injected with rotenone or vehicle in the striatum. The dietary intervention started after full induction of motor symptoms. The effects of dietary intervention and oral treatment with different doses of levodopa were assessed weekly. Motor and cognitive functions were tested, intestinal transit was analyzed and histological examination of the brain and the colon was assessed. Our results confirm our previous findings that rotenone-induced motor and non-motor problems were alleviated by the Active diet (AD). Levodopa showed an additive beneficial effect on rotarod performance in rotenone-treated animals fed with the AD. No negative interaction effects were found between the AD and levodopa. Our findings suggest that the dietary intervention might confer additional clinical benefits on patients receiving levodopa treatment.

Promising Effects of Neurorestorative Diets on Motor, Cognitive, and Gastrointestinal Dysfunction after Symptom Development in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic nigrostriatal neurons, with reductions in the function and amount of dopaminergic synapses. Therefore, synapse loss and membrane-related pathology provide relevant targets for interventions in PD. We previously showed the beneficial preventive effects of a dietary intervention containing uridine and DHA, two precursors for membrane synthesis, in the intrastriatal rotenone model for PD. Here, we examined the therapeutic potential of the same dietary intervention on motor, cognitive, and gastrointestinal symptoms. In addition, we tested the effects of an extended nutritional formula based on the same precursors plus other nutrients that increase membrane phospholipid synthesis as well as prebiotic fibers. C57BL/6J mice received a unilateral rotenone injection in the striatum. Dietary interventions started 28 days after surgery, when motor-symptoms had developed. Readout parameters included behavioral tasks measuring motor function and spatial memory as well as intestinal function and histological examination of brain and gut to assess PD-like pathology. Our results show that rotenone-induced motor and non-motor problems were partially alleviated by the therapeutic dietary interventions providing uridine and DHA. The extended nutritional intervention containing both precursors and other nutrients that increase phospholipid synthesis as well as prebiotic fibers was more effective in normalizing rotenone-induced motor and non-motor abnormalities. The latter diet also restored striatal DAT levels, indicating its neurorestorative properties. This is the first study demonstrating beneficial effects of specific dietary interventions, given after full development of symptoms, on a broad spectrum of motor and non-motor symptoms in a mouse model for PD.

A Specific Nutrient Combination Attenuates the Reduced Expression of PSD-95 in the Proximal Dendrites of Hippocampal Cell Body Layers in a Mouse Model of Phenylketonuria.

The inherited metabolic disease phenylketonuria (PKU) is characterized by increased concentrations of phenylalanine in the blood and brain, and as a consequence neurotransmitter metabolism, white matter, and synapse functioning are affected. A specific nutrient combination (SNC) has been shown to improve synapse formation, morphology and function. This could become an interesting new nutritional approach for PKU. To assess whether treatment with SNC can affect synapses, we treated PKU mice with SNC or an isocaloric control diet and wild-type (WT) mice with an isocaloric control for 12 weeks, starting at postnatal day 31. Immunostaining for post-synaptic density protein 95 (PSD-95), a post-synaptic density marker, was carried out in the hippocampus, striatum and prefrontal cortex. Compared to WT mice on normal chow without SNC, PKU mice on the isocaloric control showed a significant reduction in PSD-95 expression in the hippocampus, specifically in the granular cell layer of the dentate gyrus, with a similar trend seen in the cornus ammonis 1 (CA1) and cornus ammonis 3 (CA3) pyramidal cell layer. No differences were found in the striatum or prefrontal cortex. PKU mice on a diet supplemented with SNC showed improved expression of PSD-95 in the hippocampus. This study gives the first indication that SNC supplementation has a positive effect on hippocampal synaptic deficits in PKU mice.

Minocycline effects on the cerebrospinal fluid proteome of experimental autoimmune encephalomyelitis rats.

To identify response biomarkers for pharmaceutical treatment of multiple sclerosis, we induced experimental autoimmune encephalomyelitis (EAE) in rats and treated symptomatic animals with minocycline. Cerebrospinal fluid (CSF) samples were collected 14 days after EAE induction at the peak of neurological symptoms, and proteomics analysis was performed using nano-LC-Orbitrap mass spectrometry. Additionally, the minocycline concentration in CSF was determined using quantitative matrix-assisted laser desorption/ionization-triple-quadrupole tandem mass spectrometry (MALDI-MS/MS) in the selected reaction monitoring (SRM) mode. Fifty percent of the minocycline-treated EAE animals did not show neurological symptoms on day 14 ("responders"), while the other half displayed neurological symptoms ("nonresponders"), indicating that minocycline delayed disease onset and attenuated disease severity in some, but not all, animals. Neither CSF nor plasma minocycline concentrations correlated with the onset of symptoms or disease severity. Analysis of the proteomics data resulted in a list of 20 differentially abundant proteins between the untreated animals and the responder group of animals. Two of these proteins, complement C3 and carboxypeptidase B2, were validated by quantitative LC-MS/MS in the SRM mode. Differences in the CSF proteome between untreated EAE animals and minocycline-treated responders were similar to the differences between minocycline-treated responders and nonresponders (70% overlap). Six proteins that remained unchanged in the minocycline-treated animals but were elevated in untreated EAE animals may be related to the mechanism of action of minocycline.

Metabolomics of cerebrospinal fluid reveals changes in the central nervous system metabolism in a rat model of multiple sclerosis.

Experimental Autoimmune Encephalomyelitis (EAE) is the most commonly used animal model for Multiple Sclerosis (MScl). CSF metabolomics in an acute EAE rat model was investigated using targetted LC-MS and GC-MS. Acute EAE in Lewis rats was induced by co-injection of Myelin Basic Protein with Complete Freund's Adjuvant. CSF samples were collected at two time points: 10 days after inoculation, which was during the onset of the disease, and 14 days after inoculation, which was during the peak of the disease. The obtained metabolite profiles from the two time points of EAE development show profound differences between onset and the peak of the disease, suggesting significant changes in CNS metabolism over the course of MBP-induced neuroinflammation. Around the onset of EAE the metabolome profile shows significant decreases in arginine, alanine and branched amino acid levels, relative to controls. At the peak of the disease, significant increases in concentrations of multiple metabolites are observed, including glutamine, O-phosphoethanolamine, branched-chain amino acids and putrescine. Observed changes in metabolite levels suggest profound changes in CNS metabolism over the course of EAE. Affected pathways include nitric oxide synthesis, altered energy metabolism, polyamine synthesis and levels of endogenous antioxidants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0306-3) contains supplementary material, which is available to authorized users.

Simultaneous analysis of plasma and CSF by NMR and hierarchical models fusion.

Because cerebrospinal fluid (CSF) is the biofluid which interacts most closely with the central nervous system, it holds promise as a reporter of neurological disease, for example multiple sclerosis (MScl). To characterize the metabolomics profile of neuroinflammatory aspects of this disease we studied an animal model of MScl-experimental autoimmune/allergic encephalomyelitis (EAE). Because CSF also exchanges metabolites with blood via the blood-brain barrier, malfunctions occurring in the CNS may be reflected in the biochemical composition of blood plasma. The combination of blood plasma and CSF provides more complete information about the disease. Both biofluids can be studied by use of NMR spectroscopy. It is then necessary to perform combined analysis of the two different datasets. Mid-level data fusion was therefore applied to blood plasma and CSF datasets. First, relevant information was extracted from each biofluid dataset by use of linear support vector machine recursive feature elimination. The selected variables from each dataset were concatenated for joint analysis by partial least squares discriminant analysis (PLS-DA). The combined metabolomics information from plasma and CSF enables more efficient and reliable discrimination of the onset of EAE. Second, we introduced hierarchical models fusion, in which previously developed PLS-DA models are hierarchically combined. We show that this approach enables neuroinflamed rats (even on the day of onset) to be distinguished from either healthy or peripherally inflamed rats. Moreover, progression of EAE can be investigated because the model separates the onset and peak of the disease.

Profiling and identification of cerebrospinal fluid proteins in a rat EAE model of multiple sclerosis.

The experimental autoimmune encephalomyelitis (EAE) model resembles certain aspects of multiple sclerosis (MScl), with common features such as motor dysfunction, axonal degradation, and infiltration of T-cells. We studied the cerebrospinal fluid (CSF) proteome in the EAE rat model to identify proteomic changes relevant for MScl disease pathology. EAE was induced in male Lewis rats by injection of myelin basic protein (MBP) together with complete Freund's adjuvant (CFA). An inflammatory control group was injected with CFA alone, and a nontreated group served as healthy control. CSF was collected at day 10 and 14 after immunization and analyzed by bottom-up proteomics on Orbitrap LC-MS and QTOF LC-MS platforms in two independent laboratories. By combining results, 44 proteins were discovered to be significantly increased in EAE animals compared to both control groups, 25 of which have not been mentioned in relation to the EAE model before. Lysozyme C1, fetuin B, T-kininogen, serum paraoxonase/arylesterase 1, glutathione peroxidase 3, complement C3, and afamin are among the proteins significantly elevated in this rat EAE model. Two proteins, afamin and complement C3, were validated in an independent sample set using quantitative selected reaction monitoring mass spectrometry. The molecular weights of the identified differentially abundant proteins indicated an increased transport across the blood-brain barrier (BBB) at the peak of the disease, caused by an increase in BBB permeability.

NMR and pattern recognition can distinguish neuroinflammation and peripheral inflammation.

Multiple Sclerosis (MScl) is a neurodegenerative disease of the CNS, associated with chronic neuroinflammation. Cerebrospinal fluid (CSF), being in closest interaction with CNS, was used to profile neuroinflammation to discover disease-specific markers. We used the commonly accepted animal model for the neuroinflammatory aspect of MScl: the experimental autoimmune/allergic encephalomyelitis (EAE). A combination of advanced (1)H NMR spectroscopy and pattern recognition methods was used to establish the metabolic profile of CSF of EAE-affected rats (representing neuroinflammation) and of two control groups (healthy and peripherally inflamed) to detect specific markers for early neuroinflammation. We found that the CSF metabolic profile for neuroinflammation is distinct from healthy and peripheral inflammation and characterized by changes in concentrations of metabolites such as creatine, arginine, and lysine. Using these disease-specific markers, we were able to detect early stage neuroinflammation, with high accuracy in a second independent set of animals. This confirms the predictive value of these markers. These findings from the EAE model may help to develop a molecular diagnosis for the early stage MScl in humans.

Fusion of metabolomics and proteomics data for biomarkers discovery: case study on the experimental autoimmune encephalomyelitis.

BACKGROUND: Analysis of Cerebrospinal Fluid (CSF) samples holds great promise to diagnose neurological pathologies and gain insight into the molecular background of these pathologies. Proteomics and metabolomics methods provide invaluable information on the biomolecular content of CSF and thereby on the possible status of the central nervous system, including neurological pathologies. The combined information provides a more complete description of CSF content. Extracting the full combined information requires a combined analysis of different datasets i.e. fusion of the data. RESULTS: A novel fusion method is presented and applied to proteomics and metabolomics data from a pre-clinical model of multiple sclerosis: an Experimental Autoimmune Encephalomyelitis (EAE) model in rats. The method follows a mid-level fusion architecture. The relevant information is extracted per platform using extended canonical variates analysis. The results are subsequently merged in order to be analyzed jointly. We find that the combined proteome and metabolome data allow for the efficient and reliable discrimination between healthy, peripherally inflamed rats, and rats at the onset of the EAE. The predicted accuracy reaches 89% on a test set. The important variables (metabolites and proteins) in this model are known to be linked to EAE and/or multiple sclerosis. CONCLUSIONS: Fusion of proteomics and metabolomics data is possible. The main issues of high-dimensionality and missing values are overcome. The outcome leads to higher accuracy in prediction and more exhaustive description of the disease profile. The biological interpretation of the involved variables validates our fusion approach.

The experimental autoimmune encephalomyelitis model for proteomic biomarker studies: from rat to human.

Multiple sclerosis (MScl) is defined by central nervous system (CNS) inflammation, demyelination and axonal damage. Some of the disease mechanisms are known but the cause of this complex disorder stays an enigma. Experimental autoimmune encephalomyelitis (EAE) is an animal model mimicking many aspects of MScl. This review aims to provide an overview over proteomic biomarker studies in the EAE model emphasizing the translational aspects with respect to MScl in humans.

Two distinct classes of novel pyrazolinecarboxamides as potent cannabinoid CB1 receptor agonists.

The synthesis and SAR of 3-alkyl-4-aryl-4,5-dihydropyrazole-1-carboxamides 1-23 and 1-alkyl-5-aryl-4,5-dihydropyrazole-3-carboxamides 24-27 as two novel cannabinoid CB(1) receptor agonist classes were described. The target compounds elicited high affinities to the CB(1) as well as the CB(2) receptor and were found to act as CB(1) receptor agonists. The key compound 19 elicited potent CB(1) agonistic and CB(2) inverse agonistic properties in vitro and showed in vivo activity in a rodent model for multiple sclerosis after oral administration.