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Sickle cell disease (SCD) is an inherited red blood cell disorder that occurs worldwide. Acute vaso-occlusive crisis is the main cause of hospitalization in patients with SCD. There is growing evidence that inflammatory vasculopathy plays a key role in both acute and chronic SCD-related clinical manifestations. In a humanized mouse model of SCD, we found an increase of von Willebrand factor activity and a reduction in the ratio of a disintegrin and metalloproteinase with thrombospondin type 1 motif, number 13 (ADAMTS13) to von Willebrand factor activity similar to that observed in the human counterpart. Recombinant ADAMTS13 was administered to humanized SCD mice before they were subjected to hypoxia/reoxygenation (H/R) stress as a model of vaso-occlusive crisis. In SCD mice, recombinant ADAMTS13 reduced H/R-induced hemolysis and systemic and local inflammation in lungs and kidneys. It also diminished H/R-induced worsening of inflammatory vasculopathy, reducing local nitric oxidase synthase expression. Collectively, our data provide for the firsttime evidence that pharmacological treatment with recombinant ADAMTS13 (TAK-755) diminished H/R-induced sickle cell-related organ damage. Thus, recombinant ADAMTS13 might be considered as a potential effective disease-modifying treatment option for sickle cell-related acute events.

Aims: We investigated N471D WASH complex subunit strumpellin (Washc5) knock-in and Washc5 knock-out mice as models for hereditary spastic paraplegia type 8 (SPG8)., Methods: We generated heterozygous and homozygous N471D Washc5 knock-in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting., Results: Homozygous N471D Washc5 knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock-in mice. WASHC5-related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock-out mice showing normal WASHC5 levels could not be bred to homozygosity., Conclusions: While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively., (© 2021 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Mammalian transglutaminases (TGs) catalyze calcium-dependent irreversible posttranslational modifications of proteins and their enzymatic activities contribute to the pathogenesis of several human neurodegenerative diseases. Although different transglutaminases are found in many different tissues, the TG6 isoform is mostly expressed in the CNS. The present study was embarked on/undertaken to investigate expression, distribution and activity of transglutaminases in Huntington disease transgenic rodent models, with a focus on analyzing the involvement of TG6 in the age- and genotype-specific pathological features relating to disease progression in HD transgenic mice and a tgHD transgenic rat model using biochemical, histological and functional assays. Our results demonstrate the physical interaction between TG6 and (mutant) huntingtin by co-immunoprecipitation analysis and the contribution of its enzymatic activity for the total aggregate load in SH-SY5Y cells. In addition, we identify that TG6 expression and activity are especially abundant in the olfactory tubercle and piriform cortex, the regions displaying the highest amount of mHTT aggregates in transgenic rodent models of HD. Furthermore, mHTT aggregates were colocalized within TG6-positive cells. These findings point towards a role of TG6 in disease pathogenesis via mHTT aggregate formation.

In our previous study, we found that prenatal trauma exposure leads to an anxiety phenotype in mouse pups, characterized by increased corticosterone levels and increased anxiety-like behavior. In order to understand the mechanisms by which aversive in utero experience leads to these long-lasting behavioral and neuroendocrine changes, we investigated stress reactivity of prenatally traumatized (PT) mice, as well as the expression and methylation levels of several key regulatory genes of the stress axis in the dorsal hippocampus (dHPC) of the PT embryo and adult mice. We detected increased corticotropin-releasing hormone receptor 1 (Crhr1) and decreased FK506 binding protein 5 (Fkbp5) mRNA levels in the left dHPC of adult PT mice. These alterations were accompanied by a decreased methylation status of the Crhr1 promoter and an increased methylation status of the Fkbp5 promoter, respectively. Interestingly, the changes in Fkbp5 and Crhr1 mRNA levels were not detected in the embryonic dHPC of PT mice. Together, our findings provide evidence that prenatal trauma has a long-term impact on stress axis function and anxiety phenotype associated with altered Crhr1 and Fkbp5 transcripts and promoter methylation.

Gadolinium based contrast agents (GBCAs) are widely used in clinical MRI since the mid-1980s. Recently, concerns have been raised that trace amounts of Gadolinium (Gd), detected in brains even long time after GBCA application, may cause yet unrecognized clinical consequences. We therefore assessed the behavioral phenotype, neuro-histopathology, and Gd localization after repeated administration of linear (gadodiamide) or macrocyclic (gadobutrol) GBCA in rats. While most behavioral tests revealed no difference between treatment groups, we observed a transient and reversible decrease of the startle reflex after gadodiamide application. Residual Gd in the lateral cerebellar nucleus was neither associated with a general gene expression pathway deregulation nor with neuronal cell loss, but in gadodiamide-treated rats Gd was associated with the perineuronal net protein aggrecan and segregated to high molecular weight fractions. Our behavioral finding together with Gd distribution and speciation support a substance class difference for Gd presence in the brain after GBCA application.

Antipsychotic drugs are effective interventions in schizophrenia. However, the efficacy of these agents often decreases over time, which leads to treatment failure and symptom recurrence. We report that antipsychotic efficacy in rat models declines in concert with extracellular striatal dopamine levels rather than insufficient dopamine D2 receptor occupancy. Antipsychotic efficacy was associated with a suppression of dopamine transporter activity, which was reversed during failure. Antipsychotic failure coincided with reduced dopamine neuron firing, which was not observed during antipsychotic efficacy. Synaptic field responses in dopamine target areas declined during antipsychotic efficacy and showed potentiation during failure. Antipsychotics blocked synaptic vesicle release during efficacy but enhanced this release during failure. We found that the pharmacological inhibition of the dopamine transporter rescued antipsychotic drug treatment outcomes, supporting the hypothesis that the dopamine transporter is a main target of antipsychotic drugs and predicting that dopamine transporter blockers may be an adjunct treatment to reverse antipsychotic treatment failure.

Gait analysis of transgenic mice and rats modeling human diseases often suffers from the condition that those models exhibit genotype-driven differences in body size, weight, and length. Thus, we hypothesized that scaling by the silhouette length improves the reliability of gait analysis allowing normalization for individual body size differences. Here, we computed video-derived silhouette length and area parameters from a standard markerless gait analysis system using image-processing techniques. By using length- and area-derived data along with body weight and age, we systematically scaled individual gait parameters. We compared these different scaling approaches and report here that normalization for silhouette length improves the validity and reliability of gait analysis in general. The application of this silhouette length scaling to transgenic Huntington disease mice and Parkinson´s disease rats identifies the remaining differences reflecting more reliable, body length-independent motor functional differences. Overall, this emphasizes the need for silhouette-length-based intra-assay scaling as an improved standard approach in rodent gait analysis., (Copyright © 2019 Timotius et al.)

Background: Motor impairment appears as a characteristic symptom of several diseases and injuries. Therefore, tests for analyzing motor dysfunction are widely applied across preclinical models and disease stages. Among those, gait analysis tests are commonly used, but they generate a huge number of gait parameters. Thus, complications in data analysis and reporting raise, which often leads to premature parameter selection., New Methods: In order to avoid arbitrary parameter selection, we present here a systematic initial data analysis by utilizing heat-maps for data reporting. We exemplified this approach within an intervention study, as well as applied it to two longitudinal studies in rodent models related to Parkinson's disease (PD) and Huntington disease (HD)., Results: The systematic initial data analysis (IDA) is feasible for exploring gait parameters, both in experimental and longitudinal studies. The resulting heat maps provided a visualization of gait parameters within a single chart, highlighting important clusters of differences., Comparison With Existing Method: Often, premature parameter selection is practiced, lacking comprehensiveness. Researchers often use multiple separated graphs on distinct gait parameters for reporting. Additionally, negative results are often not reported., Conclusions: Heat mapping utilized in initial data analysis is advantageous for reporting clustered gait parameter differences in one single chart and improves data mining., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gait and postural control dysfunction are prototypical symptoms compromising quality of life for patients with Parkinson's disease (PD). Hallmarks of cellular pathology are dopaminergic degeneration and accumulation of the cytosolic protein alpha-synuclein, linked to impaired autophagy-lysosome pathway (ALP) clearance. Physical exercise improves gait in PD patients and motor function in rodent lesion models. Moreover, exercise is considered neuroprotective and ALP induction has been reported, e.g. in human skeletal muscle, rodent peripheral and cerebral tissues. A combined analysis of how distinct exercise paradigms affect motor and central biochemical aspects of PD could maximize benefits for patients. Here we examine the effect of 4 weeks treadmill exercise intervention in 7-8 month non-lesioned mice on a) distinct gait categories, b) ALP activity, c) dopaminergic and alpha-synuclein homeostasis. The study includes wild type, alpha-synuclein knockout, and mice exclusively expressing human alpha-synuclein. Parameters of gait regularity and stability, activity, and dynamic postural control during unforced walk, were assessed by an automated system (CatWalk XT). At baseline, alpha-synuclein mouse models exhibited irregular and less active gait, with impaired dynamic postural control, compared to wild type mice. Treadmill exercise particularly improved speed and stride length, while increasing dual diagonal versus three-paw body support in both the alpha-synuclein knockout and transgenic mice. Biochemical analyses showed higher striatal tyrosine hydroxylase immuno-reactivity and reduced higher-order alpha-synuclein species in the cerebral cortex. However, no significant cerebral ALP induction was measured. In summary, treadmill exercise improved gait activity and postural stability, and promoted dopaminergic and alpha-synuclein homeostasis, without robustly inducing cerebral ALP., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Dipeptidyl peptidase 4 (DPP4, CD26) is a moonlighting enzyme responsible for the proteolytic inactivation of neuropeptide Y (NPY), a peptide known for its anxiolytic effect in the central nervous system. Our previous work revealed a stress-resilient phenotype and a potentiation of short-term fear extinction in a congenic rat model deficient for DPP4 activity (DPP4mut). Here, we investigated neuroendocrine mechanisms underlying the phenotype of the DPP4mut animals. We studied the function of the hypothalamus-pituitary-adrenal (HPA) axis including the expression levels of its key genes and explored the possibility of structural NPY system changes., Methods and Results: We find decreased expression of Nr3c1 (glucocorticoid receptor - GR) and Fkbp5 (FK506 binding protein 5) in the amygdala and the hypothalamus of the DPP4mut rats, as well as the lower stress-induced peripheral corticosterone (CORT) levels. We detect no significant alterations in basal and DEX-induced CORT levels in the DPP4mut animals. The abundance of NPY-ergic neurons in the basolateral amygdala, dentate gyrus and hippocampus did not differ between the DPP4mut and their wild type littermates., Conclusion: DPP4mut rats show blunted CORT response in line with their lower behavioral stress-response profile. These results are consistent with the hypothesis that increased central NPY levels elevate the threshold of stress response. We suggest that changes in the expression levels of key HPA axis genes (Nr3c1 and Fkbp5) are a consequence of the altered stress-perception of DPP4mut animals, thus further contributing to the stress-resilient phenotype., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by expanded CAG repeats in the huntingtin gene ( HTT ). Although mutant HTT is expressed during embryonic development and throughout life, clinical HD usually manifests later in adulthood. A number of studies document neurodevelopmental changes associated with mutant HTT , but whether these are reversible under therapy remains unclear. Here, we identify very early behavioral, molecular, and cellular changes in preweaning transgenic HD rats and mice. Reduced ultrasonic vocalization, loss of prepulse inhibition, and increased risk taking are accompanied by disturbances of dopaminergic regulation in vivo, reduced neuronal differentiation capacity in subventricular zone stem/progenitor cells, and impaired neuronal and oligodendrocyte differentiation of mouse embryo-derived neural stem cells in vitro. Interventional treatment of this early phenotype with the histone deacetylase inhibitor (HDACi) LBH589 led to significant improvement in behavioral changes and markers of dopaminergic neurotransmission and complete reversal of aberrant neuronal differentiation in vitro and in vivo. Our data support the notion that neurodevelopmental changes contribute to the prodromal phase of HD and that early, presymptomatic intervention using HDACi may represent a promising novel treatment approach for HD., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Background: Sway is a crucial gait characteristic tightly correlated with the risk of falling in patients with Parkinsońs disease (PD). So far, the swaying pattern during locomotion has not been investigated in rodent models using the analysis of dynamic footprint recording obtained from the CatWalk gait recording and analysis system., New Methods: We present three methods for describing locomotion sway and apply them to footprint recordings taken from C57BL6/N wild-type mice and two different α-synuclein transgenic PD-relevant mouse models (α-syn m -ko, α-syn m -koxα-syn h -tg). Individual locomotion data were subjected to three different signal processing analytical approaches: the first two methods are based on Fast Fourier Transform (FFT), while the third method uses Low Pass Filters (LPF). These methods use the information associated with the locomotion sway and generate sway-related parameters., Results: The three proposed methods were successfully applied to the footprint recordings taken from all paws as well as from front/hind-paws separately. Nine resulting sway-related parameters were generated and successfully applied to differentiate between the mouse models under study. Namely, α-synucleinopathic mice revealed higher sway and sway itself was significantly higher in the α-syn m -koxα-syn h -tg mice compared to their wild-type littermates in eight of the nine sway-related parameters., Comparison With Existing Method: Previous locomotion sway index computation is based on the estimated center of mass position of mice., Conclusions: The methods presented in this study provide a sway-related gait characterization. Their application is straightforward and may lead to the identification of gait pattern derived biomarkers in rodent models of PD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The transgenic rat model of Huntington disease expressing a fragment of mutant HTT (tgHD rat) has been thoroughly characterized and reproduces hallmark symptoms of human adult-onset HD. Pursuing the optimization of this model for evaluation of translational therapeutic approaches, the F344 inbred rat strain was considered as advantageous genetic background for the expression of the HD transgenic construct. In the present study, a novel congenic line of the SPRDtgHD transgenic model of HD, carrying 51 CAG repeats, was generated on the F344 rat genetic background. To assess the behavioral phenotype, classical assays investigating motor function, emotion, and sensorimotor gating were applied, along with automated screening of metabolic and activity parameters as well as operant conditioning tasks. The neuropathological phenotype was analyzed by immunohistochemistry and ex vivo magnetic resonance imaging. F344tgHD rats displayed markedly reduced anxiety-like behavior in the social interaction test and elevated impulsivity traits already at 3 months of age. Neuropathologically, reduced striatal volume and pronounced aggregation of mutant huntingtin in several brain regions were detected at later disease stage. In conclusion, the congenic F344tgHD model reproduces key aspects of the human HD phenotype, substantiating its value for translational therapeutic approaches.

Characterizing gait is important in the study of movement disorders, also in clinical mouse models. Gait data are therefore necessary for the development of gait analysis methods and the study of diseases. This article presents gait data of two α-synucleinopathic transgenic mouse models and their non-transgenic littermate, backcrossed into the C57BL/6N genetic background. The animal gait was recorded using CatWalk system, which provides the information for each run about the paw positions, paw print sizes, and paw intensities as a function of time or video frame. A total of 90 run data files are provided in this article.

Rationale: The polyglutamine disease spinocerebellar ataxia type 17 (SCA17) is a neurodegenerative disease leading to severe neurological symptoms during development. Additionally, patients affected by SCA17 display psychosis earlier than their motor disorders., Objective: Here the putative psychotic phenotype and endophenotype of transgenic SCA17 rats was examined., Methods: The expression of schizophrenia-like symptoms was evaluated over a longitudinal period before and after the onset of neurological symptoms in SCA17. To this end, transgenic SCA17 rats' monoamine neurotransmission was investigated along with their locomotion at baseline and in response to amphetamine using in-vivo microdialysis in free moving conditions, their sensorimotor gating using pre-pulse inhibition of startle reaction, and their object memory using the novel object recognition test as an index of cognitive impairments., Results: Presymptomatic SCA17 rats displayed dysregulated monoamine levels at baseline and in response to amphetamine compared with control wild-type (wt) rats. At that stage, neither amphetamine-induced hyperlocomotion nor sensorimotor gating differed from that in wt rats. Symptomatic SCA17 rats developed sensorimotor gating deficits and also showed an impaired object memory, while their monoaminergic responses remained supersensitive to amphetamine., Conclusions: The data of the present study demonstrate a neurochemical endophenotype in SCA17 rats resembling that of prodromal schizophrenia. These findings suggest that a sensitization of the monoamine systems arises early in adulthood in SCA17 rats and may predispose them to express schizophrenia-like symptoms later in life.

Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age-related and brain region-specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP-transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP-transgenic mouse and one APP-transgenic rat model. We observed remarkable differences in expression levels and brain region-specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP-transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals., (© 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the ε-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Inhibitors of dipeptidyl peptidase 4 (DPP4, CD26) are used for the treatment of type 2 diabetic patients and better glucose tolerance has been confirmed in functionally DPP4-deficient congenic rats (DPP4mut), along with immunological alterations and, interestingly, a stress-resilient phenotype. All these findings are in agreement with the "moonlighting" properties of DPP4, whose proteolytic action is responsible for the inactivation of a number of regulatory peptides including, but not limited to, neuropeptide Y (NPY). Among all candidate substrates, DPP4 displays highest affinity for NPY, an endogenous anxiolytic neurotransmitter that is suggested as a candidate biomarker in post-traumatic stress disorder (PTSD) and depression., Methods and Results: Central and peripheral NPY levels were measured by ELISA in DPP4mut and DAwt rats revealing a significantly higher concentration of the peptide in the CSF of DPP4mut animals. This finding positively correlated with the blunted stress phenotype measured on an analgesia-meter. Additionally, when a classical fear-conditioning paradigm was investigated, short-term fear extinction was significantly potentiated in DPP4mut rats as compared to wt controls., Conclusions: Our findings indicate a positive correlation between reduced stress-responsiveness and increased central NPY, in DPP4mut rats. Most interestingly, the behavioral phenotype extends to facilitation of fear extinction. These observations raise further interest in DPP4-modulating drugs for the potential effect on NPY metabolism, as a therapeutic tool for psychiatric conditions such as anxiety disorders and PTSD., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The need for improving throughput, validity, and reliability in the behavioral characterization of rodents may benefit from integrating automated intra-home-cage-screening systems allowing the simultaneous detection of multiple behavioral and physiological parameters in parallel., New Method: To test this hypothesis, transgenic Huntington's disease (tgHD) rats were repeatedly screened within phenotyping home-cages (PhenoMaster and IntelliCage for rats), where spontaneous activity, feeding, drinking, temperature, and metabolic performance were continuously measured. Cognition and emotionality were evaluated within the same environment by means of operant learning procedures and refined analysis of the behavioral display under conditions of novelty. This investigator-independent approach was further correlated with behavioral display of the animals in classical behavioral assays. Multivariate analysis (MVA) including Principle Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) was used to explore correlation patterns of variables within and across the two genotypes., Results: The automated systems traced previously undetected aspects in the phenotype of tgHD rats (circadian activity, energy metabolism, rearing), and out of those spontaneous free rearing correlated with individual performance in the accelerod test. PCA revealed a segregation by genotype in juvenile tgHD rats that differed from adult animals, being further resolved by PLS-DA detecting "temperature" (juvenile) and "rearing" (adult) as phenotypic key variables in the tgHD model., Conclusions: Intra-home-cage phenotyping in combination with MVA, is capable of characterizing a complex phenotype by detecting novel physiological and behavioral markers with high sensitivity and standardization using fewer human resources. A broader application of automated systems for large-scale screening is encouraged., (Copyright © 2014. Published by Elsevier B.V.)

Background: Posttranslational modifications of beta amyloid (Aβ) have been shown to affect its biophysical and neurophysiological properties. One of these modifications is N-terminal pyroglutamate (pE) formation. Enzymatic glutaminyl cyclase (QC) activity catalyzes cyclization of truncated Aβ(3-x), generating pE3-Aβ. Compared to unmodified Aβ, pE3-Aβ is more hydrophobic and neurotoxic. In addition, it accelerates aggregation of other Aβ species. To directly investigate pE3-Aβ formation and toxicity in vivo, transgenic (tg) ETNA (E at the truncated N-terminus of Aβ) mice expressing truncated human Aβ(3-42) were generated and comprehensively characterized. To further investigate the role of QC in pE3-Aβ formation in vivo, ETNA mice were intercrossed with tg mice overexpressing human QC (hQC) to generate double tg ETNA-hQC mice., Results: Expression of truncated Aβ(3-42) was detected mainly in the lateral striatum of ETNA mice, leading to progressive accumulation of pE3-Aβ. This ultimately resulted in astrocytosis, loss of DARPP-32 immunoreactivity, and neuronal loss at the sites of pE3-Aβ formation. Neuropathology in ETNA mice was associated with behavioral alterations. In particular, hyperactivity and impaired acoustic sensorimotor gating were detected. Double tg ETNA-hQC mice showed similar Aβ levels and expression sites, while pE3-Aβ were significantly increased, entailing increased astrocytosis and neuronal loss., Conclusions: ETNA and ETNA-hQC mice represent novel mouse models for QC-mediated toxicity of truncated and pE-modified Aβ. Due to their significant striatal neurodegeneration these mice can also be used for analysis of striatal regulation of basal locomotor activity and sensorimotor gating, and possibly for DARPP-32-dependent neurophysiology and neuropathology. The spatio-temporal correlation of pE3-Aβ and neuropathology strongly argues for an important role of this Aβ species in neurodegenerative processes in these models.

Background: As a key enzyme in sphingolipid metabolism, acid sphingomyelinase (ASM) is involved in the regulation of cell fate and signaling via hydrolysis of sphingomyelin to form ceramide. While increased activity of the lysosomal form has been associated with various pathological conditions, there are few studies on secretory ASM limited only to cell models, plasma or serum., Methods: An optimized assay based on a fluorescent substrate was applied to measure the ASM activity in cerebrospinal fluid (CSF) collected from mice and from 42 patients who were classified as controls based on normal routine CSF values., Results: We have detected ASM activity in human CSF, established a sensitive quantitative assay and characterized the enzyme's properties. The enzyme resembles plasmatic ASM including protein stability and Zn(2+)-dependence but the assays differ considerably in the optimal detergent concentration. Significantly increased activities in the CSF of ASM transgenic mice and undetectable levels in ASM knock-out mice prove that the measured ASM activity originates from the ASM-encoding gene SMPD1. CSF localized ASM activities were comparable to corresponding serum ASM levels at their respective optimal reaction conditions, but no correlation was observed. The large variance in ASM activity was independent of sex, age or analyzed routine CSF parameters., Conclusions: Human and mouse CSF contain detectable levels of secretory ASM, which are unrelated to serum ASM activities. Further investigations in humans and in animal models will help to elucidate the role of this enzyme in human disease and to assess its value as a potential biomarker for disease type, severity, progress or therapeutic success.

The care of frail and dependent elderly people living at home has become a major healthcare issue. Following a multi-disciplinary approach, the gerontology healthcare network Giront'Emeraude in Saint-Malo offers support within the local community. Trained by the network, nurses play a pivotal role in the coordination of the personalised care projects of the elderly people within the network.

Huntington's disease (HD) is a neurodegenerative disorder, which is characterized by progressive motor impairment and cognitive alterations. Changes in energy metabolism, neuroendocrine function, body weight, euglycemia, appetite function, and circadian rhythm can also occur. It is likely that the locus of these alterations is the hypothalamus. We used the HD transgenic (tg) rat model bearing 51 CAG repeats, which exhibits similar HD symptomology as HD patients to investigate hypothalamic function. We conducted detailed hypothalamic proteome analyses and also measured circulating levels of various metabolic hormones and lipids in pre-symptomatic and symptomatic animals. Our results demonstrate that there are significant alterations in HD rat hypothalamic protein expression such as glial fibrillary acidic protein (GFAP), heat shock protein-70, the oxidative damage protein glutathione peroxidase (Gpx4), glycogen synthase1 (Gys1) and the lipid synthesis enzyme acylglycerol-3-phosphate O-acyltransferase 1 (Agpat1). In addition, there are significant alterations in various circulating metabolic hormones and lipids in pre-symptomatic animals including, insulin, leptin, triglycerides and HDL, before any motor or cognitive alterations are apparent. These early metabolic and lipid alterations are likely prodromal signs of hypothalamic dysfunction. Gaining a greater understanding of the hypothalamic and metabolic alterations that occur in HD, could lead to the development of novel therapeutics for early interventional treatment of HD.

RACK1 (Receptor for Activated C Kinase 1) is a scaffold protein for different kinases and membrane receptors. Previously, we characterized an age-dependent decline of RACK1 protein expression which could be counteracted with DHEA (dehydroepiandrosterone) [Corsini, E., et al. 2002. In vivo dehydroepiandrosterone restores age-associated defects in the protein kinase C signal transduction pathway and related functional responses. J. Immunol. 168, 1753-1758. and Corsini, E., et al. 2005. Age-related decline in RACK-1 expression in human leukocytes is correlated to plasma levels of dehydroepiandrosterone. J. Leukoc. Biol. 77, 247-256.]. Hypothesizing a direct control of RACK1 expression by DHEA we studied the not yet characterized human promoter region of its coding gene GNB2L1. The FLOE (Fluorescently Labeled Oligonucleotide Extension) was used to map the transcription start site and a novel Gateway luciferase vector (GW luc basic; Del Vecchio, I., Zuccotti, A., Canneva, F., Lenzken, S.C., Racchi, M., 2007. Development of the first Gateway firefly luciferase vector and use of reverse transcriptase in FLOE (Fluorescently Labeled Oligonucleotide Extension) reactions. Plasmid 58, 269-274.) to obtain promoter region mutants. Human SH-SY5Y, THP1 and lymphoblastoid cells were used for transient transfections and treatments with lipopolysaccharide (LPS), phorbol myristate acetate (PMA), DHEA and cortisol (the first two molecules to differently activate NF-kB, a transcription complex able to regulate the murine Gnb2l1 gene expression, whereas DHEA and cortisol since they are known to be imbalanced during the aging and possess counteracting actions on the immune function). The primer extension demonstrated the existence of two alternative start sites of transcription respectively located at about 230 and 300 nt 5' of the Genbank mRNA entry for GNB2L1. Moreover, as a result of the luciferase study we were able to demonstrate that a little region of approximately 300 nt conserved sufficient elements for reporter expression. We also reported that the DHEA modulation of GNB2L1 endogenous expression could not be recapitulated with the luciferase assays. Indeed, the promoter was significantly modulated by means of LPS and PMA treatments but not using DHEA. Differently the use of cortisol led us to demonstrate a biologically significant decrease of luciferase activity only in the presence of a binding site for nuclear receptors of glucocorticoids. Interestingly, other binding sites for transcriptional factors were identified in silico: different c-Rel (NF-kB) and some cardiomyocitic specific cis-acting elements. All this data suggest that the DHEA mediated GNB2L1 regulation is modulated by distant elements (enhancers/silencers), whereas LPS, PMA and cortisol effect can act directly on the mapped GNB2L1 promoter. In conclusion we hypothesize that the imbalance between DHEA and cortisol during aging could be important in the previously demonstrated recovery of the RACK1 expression.

The present commentary based on cell and animal models of intracellular beta-amyloid (iAbeta) expression indicates that low levels of microscopically undetectable iAbeta could have a physiological role in the modulation of the cyclic AMP response element (CRE)-dependent gene expression and, as a consequence, a positive influence on synaptic plasticity (the 'good' Abeta?). On the other hand, high levels of iAbeta resembling the pathological and microscopically visible accumulation of this amyloid peptide, akin to that observed in Down syndrome and Alzheimer's disease, disrupt CRE-regulated gene expression, therefore compromising the protein synthesis-dependent component of long-term potentiation (the 'bad' Abeta?). Moreover, intracellular pathology would be independent and additive to the toxic effects of the extracellular Abeta burden., (2008 S. Karger AG, Basel)

To study promoters we usually use primer extension to map the transcription start site and a panel of PCR generated deletion mutants. This strategy is complex and time-consuming. Therefore, we decided to improve it by using Gateway and FLOE (Fluorescently Labeled Oligonucleotide Extension). In this report we developed the first luciferase reporter "destination vector" (GW luc basic) for the Gateway technology and tested its efficacy, accuracy and background level by transfecting two distant cell lines (THP1 monocytic and SH-SY5Y neural cells). This vector is a real advantage for the cloning of many PCR fragments and sustains reporter activity also in THP1 cells, which are known to be problematic for transfection/expression. FLOE is a straightforward method to map transcription start sites but a bias in the capillary electrophoretic migration pattern of ROX weight markers has been reported: ROX markers migrated as if they were some bp longer. We hypothesized that this could depend on the use of different enzymes for the two principal reactions (DNA polymerase for the dideoxy chain terminated reaction on DNA and reverse transcriptase for the primer extension on RNA). Therefore, we used the same reverse transcriptase enzyme on both reactions, demonstrating that the reported bias is not due to the use of different enzymes but is an intrinsic feature of the ROX markers. The proposed procedure is important not only because of the timeliness but also for the global impact on the study of the first layer of the gene regulation.

In a previous work, we demonstrated that the Mycobacterium tuberculosis Rv2358-furB operon is induced by zinc. In this study, the orthologous genes from Mycobacterium smegmatis mc(2)155 were inactivated and mutants analyzed. Rv2358 protein was purified and found to bind upstream of the Rv2358 gene. Binding was inhibited by Zn(2+) ions.

The Mycobacterium tuberculosis genome encodes two ferric uptake regulator homologues, furA and furB, the function of which is under investigation. Using Mycobacterium smegmatis as a model system, we investigated the transcriptional pattern of Rv(Ms)2358-furB genes. Transcripts covering the two genes could be identified by northern blotting and by reverse transcriptase PCR. The transcriptional start point was mapped at one base upstream of the Ms2358 start codon by the RACE technique. By cloning M. smegmatis and M. tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of one promoter, located immediately upstream of the Rv(Ms)2358 gene. Promoter induction was tested on several cultures grown under different conditions of pH and temperature, and in the presence of different concentrations of metallic ions. The promoter was found to be specifically induced by zinc. The recombinant M. tuberculosis FurB protein typically contained two zinc ions per protein monomer. Complete removal of zinc could not be obtained, even with strong denaturation treatment. Our data are in favour of the hypothesis that Rv2358 and FurB are transcriptional regulators involved in zinc homeostasis.

The furA-katG region of Mycobacterium tuberculosis, encoding a Fur-like protein and the catalase-peroxidase, is highly conserved among mycobacteria. Both genes are induced upon oxidative stress. In this work we analyzed the M. tuberculosis furA promoter region. DNA fragments were cloned upstream of the luciferase reporter gene, and promoter activity in Mycobacterium smegmatis was measured in both the presence and absence of oxidative stress. The shortest fragment containing an inducible promoter extends 45 bp upstream of furA. In this region, -35 and -10 promoter consensus sequences can be identified, as well as a 23-bp AT-rich sequence that is conserved in the nonpathogenic but closely related M. smegmatis. M. tuberculosis FurA was purified and found to bind upstream of furA by gel shift analysis. A ca. 30-bp DNA sequence, centered on the AT-rich region, was essential for FurA binding and protected by FurA in footprinting analysis. Peroxide treatment of FurA abolished DNA binding. Three different AT-rich sequences mutagenized by site-directed mutagenesis were constructed. In each mutant, both M. tuberculosis FurA binding in vitro and pfurA regulation upon oxidative-stress in M. smegmatis were abolished. Thus, pfurA is an oxidative stress-responsive promoter controlled by the FurA protein.