To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.
Brain-derived neurotrophic factor (BDNF) and apolipoprotein E (ApoE) are thought to be implicated in a variety of neuronal processes, including cell growth, resilience to noxious stimuli and synaptic plasticity. A Val to Met substitution at codon 66 in the BDNF protein has been associated with a variety of neuropsychiatric conditions. The ApoE4 allele is considered a risk factor for late-onset Alzheimer’s disease, but its effects on young adults are less clear. We sought to investigate the effects of those two polymorphisms on hemispheric and lateral ventricular volumes of young healthy adults.
Hemispheric and lateral ventricular volumes of 144 healthy individuals, aged 19–35 years, were measured using high resolution magnetic resonance imaging and data were correlated with BDNF and ApoE genotypes.
There were no correlations between BDNF or ApoE genotype and hemispheric or lateral ventricular volumes.
These findings indicate that it is unlikely that either the BDNF Val66Met or ApoE polymorphisms exert any significant effect on hemispheric or lateral ventricular volume. However, confounding epistatic genetic effects as well as relative insensitivity of the volumetric methods used cannot be ruled out. Further imaging analyses are warranted to better define any genetic influence of the BDNF Val6Met and ApoE polymorphism on brain structure of young healthy adults.
apolipoprotein E; brain-derived neurotrophic factor; magnetic resonance imaging; polymorphism; volumetry
Rare loss-of-function mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene are known to dramatically decrease the catalytic activity of acid sphingomyelinase (ASM), resulting in an autosomal recessive lysosomal storage disorder known as Niemann-Pick disease (NPD) type A and B. In contrast to the general low frequency of those deleterious mutations, we found a relatively high frequency for the proposed type B NPD variant c.1460C>T (p.A487V) in our sample of 58 patients suffering from Major Depressive Disorder. We therefore investigated the biochemical consequences of this variant more closely. Our in vivo data derived from blood cell analyses indicated cellular ASM activity levels in the normal range. The secreted ASM activity levels in blood plasma were slightly lower, but still above those levels reported for type B NPD patients. In vitro expression studies of this ASM variant in different cell lines confirmed these results, showing cellular and secreted enzymatic activities equivalent to those of wild-type ASM and similar expression levels. Thus, we conclude that the ASM variant c.1460C>T (p.A487V) is not a rare missense mutation but an SMPD1 sequence variant that yields a protein with functional catalytic characteristics.
Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity.
We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models.
These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity.
Multivariate image analysis has shown potential for classification between Alzheimer's disease (AD) patients and healthy controls with a high-diagnostic performance. As image analysis of positron emission tomography (PET) and single photon emission computed tomography (SPECT) data critically depends on appropriate data preprocessing, the focus of this work is to investigate the impact of data preprocessing on the outcome of the analysis, and to identify an optimal data preprocessing method. In this work, technetium-99methylcysteinatedimer (99mTc-ECD) SPECT data sets of 28 AD patients and 28 asymptomatic controls were used for the analysis. For a series of different data preprocessing methods, which includes methods for spatial normalization, smoothing, and intensity normalization, multivariate image analysis based on principal component analysis (PCA) and Fisher discriminant analysis (FDA) was applied. Bootstrap resampling was used to investigate the robustness of the analysis and the classification accuracy, depending on the data preprocessing method. Depending on the combination of preprocessing methods, significant differences regarding the classification accuracy were observed. For 99mTc-ECD SPECT data, the optimal data preprocessing method in terms of robustness and classification accuracy is based on affine registration, smoothing with a Gaussian of 12 mm full width half maximum, and intensity normalization based on the 25% brightest voxels within the whole-brain region.
Alzheimer's disease (AD); intensity normalization; multivariate analysis; principal component analysis (PCA); single photon emission computed tomography (SPECT); spatial normalization
Currently available pharmacological and non-pharmacological treatments have shown only modest effects in slowing the progression of dementia. Our objective was to assess the impact of a long-term non-pharmacological group intervention on cognitive function in dementia patients and on their ability to carry out activities of daily living compared to a control group receiving the usual care.
A randomized, controlled, single-blind longitudinal trial was conducted with 98 patients (follow-up: n = 61) with primary degenerative dementia in five nursing homes in Bavaria, Germany. The highly standardized intervention consisted of motor stimulation, practice in activities of daily living, and cognitive stimulation (acronym MAKS). It was conducted in groups of ten patients led by two therapists for 2 hours, 6 days a week for 12 months. Control patients received treatment as usual. Cognitive function was assessed using the cognitive subscale of the Alzheimer's Disease Assessment Scale (ADAS-Cog), and the ability to carry out activities of daily living using the Erlangen Test of Activities of Daily Living (E-ADL test) at baseline and after 12 months.
Of the 553 individuals screened, 119 (21.5%) were eligible and 98 (17.7%) were ultimately included in the study. At 12 months, the results of the per protocol analysis (n = 61) showed that cognitive function and the ability to carry out activities of daily living had remained stable in the intervention group but had decreased in the control patients (ADAS-Cog: adjusted mean difference: -7.7, 95% CI -14.0 to -1.4, P = 0.018, Cohen's d = 0.45; E-ADL test: adjusted mean difference: 3.6, 95% CI 0.7 to 6.4, P = 0.015, Cohen's d = 0.50). The effect sizes for the intervention were greater in the subgroup of patients (n = 50) with mild to moderate disease (ADAS-Cog: Cohen's d = 0.67; E-ADL test: Cohen's d = 0.69).
A highly standardized, non-pharmacological, multicomponent group intervention conducted in a nursing-home setting was able to postpone a decline in cognitive function in dementia patients and in their ability to carry out activities of daily living for at least 12 months.
http://www.isrctn.com Identifier: ISRCTN87391496
dementia; non-pharmacological intervention; group therapy; RCT; nursing home
The prediction of blood–brain barrier permeation is vitally important for the optimization of drugs targeting the central nervous system as well as for avoiding side effects of peripheral drugs. Following a previously proposed model on blood–brain barrier penetration, we calculated the cross-sectional area perpendicular to the amphiphilic axis. We obtained a high correlation between calculated and experimental cross-sectional area (r = 0.898, n = 32). Based on these results, we examined a correlation of the calculated cross-sectional area with blood–brain barrier penetration given by logBB values. We combined various literature data sets to form a large-scale logBB dataset with 362 experimental logBB values. Quantitative models were calculated using bootstrap validated multiple linear regression. Qualitative models were built by a bootstrapped random forest algorithm. Both methods found similar descriptors such as polar surface area, pKa, logP, charges and number of positive ionisable groups to be predictive for logBB. In contrast to our initial assumption, we were not able to obtain models with the cross-sectional area chosen as relevant parameter for both approaches. Comparing those two different techniques, qualitative random forest models are better suited for blood-brain barrier permeability prediction, especially when reducing the number of descriptors and using a large dataset. A random forest prediction system (ntrees = 5) based on only four descriptors yields a validated accuracy of 88%.
Electronic supplementary material
The online version of this article (doi:10.1007/s10822-011-9478-1) contains supplementary material, which is available to authorized users.
Blood–brain barrier; Central nervous system; Membrane permeability; QSAR; LogBB; Random forest
We describe a hitherto unknown feature for 27 small drug-like molecules, namely functional inhibition of acid sphingomyelinase (ASM). These entities named FIASMAs (Functional Inhibitors of Acid SphingoMyelinAse), therefore, can be potentially used to treat diseases associated with enhanced activity of ASM, such as Alzheimer's disease, major depression, radiation- and chemotherapy-induced apoptosis and endotoxic shock syndrome. Residual activity of ASM measured in the presence of 10 µM drug concentration shows a bimodal distribution; thus the tested drugs can be classified into two groups with lower and higher inhibitory activity. All FIASMAs share distinct physicochemical properties in showing lipophilic and weakly basic properties. Hierarchical clustering of Tanimoto coefficients revealed that FIASMAs occur among drugs of various chemical scaffolds. Moreover, FIASMAs more frequently violate Lipinski's Rule-of-Five than compounds without effect on ASM. Inhibition of ASM appears to be associated with good permeability across the blood-brain barrier. In the present investigation, we developed a novel structure-property-activity relationship by using a random forest-based binary classification learner. Virtual screening revealed that only six out of 768 (0.78%) compounds of natural products functionally inhibit ASM, whereas this inhibitory activity occurs in 135 out of 2028 (6.66%) drugs licensed for medical use in humans.
The ratio of the lengths of the second and fourth finger (2D∶4D) has been
described as reflecting the degree of prenatal androgen exposure in humans.
2D∶4D is smaller for males than females and is associated with traits such
as left-handedness, physical aggression, attention-deficit-hyperactivity
disorder and a genetic polymorphism of the androgen receptor. All of these
traits are known to be correlated to the vulnerability for alcohol dependency.
We therefore hypothesized low 2D∶4D in patients with alcohol dependency.
In the present study on 131 patients suffering from alcohol dependency and 185
healthy volunteers, we found that alcohol dependent patients had smaller
2D∶4D ratios compared to controls with preserved sexual dimorphism but
with reduced right-left differences. The detection of alcohol dependency based
on 2D∶4D ratios was most accurate using the right hand of males
(ROC-analysis: AUC 0.725, sensitivity 0.667, specificity 0.723). These findings
provide novel insights into the role of prenatal androgen exposure in the
development of alcohol dependency and for the use of 2D∶4D as a possible
trait marker in identifying patients with alcohol dependency.
SPECT allows registration of regional cerebral blood flow (rCBF) which is altered in a characteristic temporoparietal pattern in Alzheimer's Dementia. Numerous studies have shown the diagnostic value of reduced cerebral blood flow and metabolic changes using perfusion SPECT and FDG-PEPT in AD diagnosis as well as in differential diagnosis against frontotemporal dementia, dementia with Lewy bodies and vascular disease. Recently more pathophysiology-based biomarkers in CSF and Amyloid-PET tracers have been developed that probably have a higher diagnostic accuracy than the more indirect rCBF changes seen in perfusion SPECT. In the paper review, we describe recent advances in AD biomarkers as well as improvements in the SPECT technique.
Late Onset Alzheimer's disease (LOAD) is the leading cause of dementia. Recent large genome-wide association studies (GWAS) identified the first strongly supported LOAD susceptibility genes since the discovery of the involvement of APOE in the early 1990s. We have now exploited these GWAS datasets to uncover key LOAD pathophysiological processes.
We applied a recently developed tool for mining GWAS data for biologically meaningful information to a LOAD GWAS dataset. The principal findings were then tested in an independent GWAS dataset.
We found a significant overrepresentation of association signals in pathways related to cholesterol metabolism and the immune response in both of the two largest genome-wide association studies for LOAD.
Processes related to cholesterol metabolism and the innate immune response have previously been implicated by pathological and epidemiological studies of Alzheimer's disease, but it has been unclear whether those findings reflected primary aetiological events or consequences of the disease process. Our independent evidence from two large studies now demonstrates that these processes are aetiologically relevant, and suggests that they may be suitable targets for novel and existing therapeutic approaches.
Animals, including humans, exhibit a variety of biological rhythms. This article describes a method for the detection and simultaneous comparison of multiple nycthemeral rhythms.
A statistical method for detecting periodic patterns in time-related data via harmonic regression is described. The method is particularly capable of detecting nycthemeral rhythms in medical data. Additionally a method for simultaneously comparing two or more periodic patterns is described, which derives from the analysis of variance (ANOVA). This method statistically confirms or rejects equality of periodic patterns. Mathematical descriptions of the detecting method and the comparing method are displayed.
Nycthemeral rhythms of incidents of bodily harm in Middle Franconia are analyzed in order to demonstrate both methods. Every day of the week showed a significant nycthemeral rhythm of bodily harm. These seven patterns of the week were compared to each other revealing only two different nycthemeral rhythms, one for Friday and Saturday and one for the other weekdays.
Synapses are distributed heterogeneously in neural networks. The relationship between the spatial arrangement of synapses and an individual synapse's structural and functional features remains to be elucidated. Here, we examined the influence of the number of adjacent synapses on individual synaptic recycling pool sizes. When measuring the discharge of the styryl dye FM1–43 from electrically stimulated synapses in rat hippocampal tissue cultures, a strong positive correlation between the number of neighbouring synapses and recycling vesicle pool sizes was observed. Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes. Although these synapses with large recycling pool sizes were rare, they were densely arranged and thus exhibited a high amount of release per volume. To consolidate these findings, functional terminals were marked by live-cell antibody staining with anti-synaptotagmin-1-cypHer or overexpression of synaptopHluorin. Analysis of synapse distributions in these systems confirmed the results obtained with FM 1–43. Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity.
The classic neuritic β-amyloid plaque of Alzheimer's disease (AD) is typically associated with activated microglia and neuroinflammation. Similarly, cerebrovascular β-amyloid (Aβ) deposits are surrounded by perivascular macrophages. Both observations indicate a contribution of the mononuclear phagocyte system to the development of β-amyloid.
Human CD14-positive mononuclear phagocytes were isolated from EDTA-anticoagulated blood by magnetic activated cell sorting. After a cultivation period of 72 hours in serum-free medium we assessed the protein levels of amyloid precursor protein (APP) as well as the patterns and the amounts of released Aβ peptides by ELISA or one-dimensional and two-dimensional urea-based SDS-PAGE followed by western immunoblotting.
We observed strong and significant increases in Aβ peptide release upon phagocytosis of acetylated low density lipoprotein (acLDL) or polystyrene beads and also after activation of the CD14/TLR4 pathway by stimulation with LPS. The proportion of released N-terminally truncated Aβ variants was increased after stimulation with polystyrene beads and acLDL but not after stimulation with LPS. Furthermore, strong shifts in the proportions of single Aβ1-40 and Aβ2-40 variants were detected resulting in a stimulus-specific Aβ signature. The increased release of Aβ peptides was accompanied by elevated levels of full length APP in the cells. The maturation state of APP was correlated with the release of N-terminally truncated Aβ peptides.
These findings indicate that mononuclear phagocytes potentially contribute to the various N-truncated Aβ variants found in AD β-amyloid plaques, especially under neuroinflammatory conditions.
We studied the diagnostic value of CSF Aβ42/tau versus low Aβ1–42% and high Aβ1–40ox% levels for differential diagnosis of Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), respectively. CSF of 45 patients with AD, 15 with DLB, 21 with Parkinson's disease dementia (PDD), and 40 nondemented disease controls (NDC) was analyzed by Aβ-SDS-PAGE/immunoblot and ELISAs (Aβ42 and tau). Aβ42/tau lacked specificity in discriminating AD from DLB and PDD. Best discriminating biomarkers were Aβ1–42% and Aβ1–40ox% for AD and DLB, respectively. AD and DLB could be differentiated by both Aβ1–42% and Aβ1–40ox% with an accuracy of 80% at minimum. Thus, we consider Aβ1–42% and Aβ1–40ox% to be useful biomarkers for AD and DLB, respectively. We propose further studies on the integration of Aβ1–42% and Aβ1–40ox% into conventional assay formats. Moreover, future studies should investigate the combination of Aβ1–40ox% and CSF alpha-synuclein for the diagnosis of DLB.
We undertook a two-stage genome-wide association study of Alzheimer's disease involving over 16,000 individuals. In stage 1 (3,941 cases and 7,848 controls), we replicated the established association with the APOE locus (most significant SNP: rs2075650, p= 1.8×10−157) and observed genome-wide significant association with SNPs at two novel loci: rs11136000 in the CLU or APOJ gene (p= 1.4×10−9) and rs3851179, a SNP 5′ to the PICALM gene (p= 1.9×10−8). Both novel associations were supported in stage 2 (2,023 cases and 2,340 controls), producing compelling evidence for association with AD in the combined dataset (rs11136000: p= 8.5×10−10, odds ratio= 0.86; rs3851179: p= 1.3×10−9, odds ratio= 0.86). We also observed more variants associated at p< 1×10−5 than expected by chance (p=7.5×10−6), including polymorphisms at the BIN1, DAB1 and CR1 loci.
Lysosomes are acidic organelles and are involved in various diseases, the most prominent is malaria. Accumulation of molecules in the cell by diffusion from the external solution into cytosol, lysosome and mitochondrium was calculated with the Fick-Nernst-Planck-equation. The cell model considers the diffusion of neutral and ionic molecules across biomembranes, dissociation to mono- or bivalent ions, adsorption to lipids, and electrical attraction or repulsion. Based on simulation results, high and selective accumulation in lysosomes was found for weak mono- and bivalent bases with intermediate to high log Kow. These findings were validated with experimental results and by a comparison to the properties of antimalarial drugs in clinical use. For ten active compounds, nine were predicted to accumulate to a greater extent in lysosomes than in other organelles, six of these were in the optimum range predicted by the model and three were close. Five of the antimalarial drugs were lipophilic weak dibasic compounds. The predicted optimum properties for a selective accumulation of weak bivalent bases in lysosomes are consistent with experimental values and are more accurate than any prior calculation. This demonstrates that the cell model can be a useful tool for the design of effective lysosome-targeting drugs with minimal off-target interactions.
accumulation; base; drug design; lysosome; malaria; model
Different classes of antidepressant drugs are used as a treatment for depression by activating the catecholinergic system. In addition, depression has been associated with decrease of growth factors, which causes insufficient axonal sprouting and reduced neuronal damage repair. In this study, antidepressant treatments are analyzed in a cell culture system, to study the modulation of growth factors.
We quantified the transcription of several growth factors in three cell lines after application of antidepressant drugs by real time polymerase chain reaction. Antidepressant drugs counteracted against phorbolester-induced deregulation of growth factors in PMA-differentiated neuronal SY5Y cells. We also found indications in a pilot experiment that magnetic stimulation could possibly modify BDNF in the cell culture system.
The antidepressant effects antidepressant drugs might be explained by selective modulation of growth factors, which subsequently affects neuronal plasticity.
Neuroleptic malignant syndrome (NMS) is a rare, but sometimes fatal, adverse reaction to neuroleptics characterized principally by fever and rigor. The aim of this study was to prove the efficacy of different NMS treatment strategies, focusing on the efficacy of dantrolene.
Altogether, 271 case reports were included. These cases were categorized into four treatment groups and compared to each other according to effectiveness of therapy within 24 hours, mortality, complete time of remission in days, effectiveness due to increase of dosage, relapse on the basis of decrease of dosage, and improvement of symptoms.
Between the four treatment groups, the complete time of remission was significantly different (analysis of variance, F = 4.02; degrees of freedom = 3; p = 0.008). In a logistic regression with adjustment for age, gender, and severity code, no significant predictor of the treatment for the complete time of remission (dichotomized by median) could be found. However, if the premedication was a monotherapy with neuroleptics, the complete time of remission was significantly shorter with dantrolene monotherapy (t = -2.97; p = 0.004).
The treatment of NMS with drugs that are combined with dantrolene is associated with a prolongation of clinical recovery. Furthermore, treatment of NMS with dantrolene as monotherapy seems to be associated with a higher overall mortality. Therefore, dantrolene does not seem to be the evidence-based treatment of choice in cases of NMS but might be useful if premedication consisted of a neuroleptic monotherapy.
Drug-induced phospholipidosis (PLD) is a lysosomal storage disorder characterized by the accumulation of phospholipids within the lysosome. This adverse drug effect can occur in various tissues and is suspected to impact cellular viability. Therefore, it is important to test chemical compounds for their potential to induce PLD during the drug design process. PLD has been reported to be a side effect of many commonly used drugs, especially those with cationic amphiphilic properties. To predict drug-induced PLD in silico, we established a high-throughput cell-culture-based method to quantitatively determine the induction of PLD by chemical compounds. Using this assay, we tested 297 drug-like compounds at two different concentrations (2.5 μm and 5.0 μm). We were able to identify 28 previously unknown PLD-inducing agents. Furthermore, our experimental results enabled the development of a binary classification model to predict PLD-inducing agents based on their molecular properties. This random forest prediction system yields a bootstrapped validated accuracy of 86 %. PLD-inducing agents overlap with those that target similar biological processes; a high degree of concordance with PLD-inducing agents was identified for cationic amphiphilic compounds, small molecules that inhibit acid sphingomyelinase, compounds that cross the blood–brain barrier, and compounds that violate Lipinski’s rule of five. Furthermore, we were able to show that PLD-inducing compounds applied in combination additively induce PLD.
cationic amphiphilic drugs; lysosomal storage disorders; phospholipidosis; phospholipids; toxicology