A human-derived antibody targets misfolded SOD1 and ameliorates motor symptoms in mouse models of amyotrophic lateral sclerosis.

Mutations in the gene encoding superoxide dismutase 1 (SOD1) lead to misfolding and aggregation of SOD1 and cause familial amyotrophic lateral sclerosis (FALS). However, the implications of wild-type SOD1 misfolding in sporadic forms of ALS (SALS) remain unclear. By screening human memory B cells from a large cohort of healthy elderly subjects, we generated a recombinant human monoclonal antibody (α-miSOD1) that selectively bound to misfolded SOD1, but not to physiological SOD1 dimers. On postmortem spinal cord sections from 121 patients with ALS, α-miSOD1 antibody identified misfolded SOD1 in a majority of cases, regardless of their SOD1 genotype. In contrast, the α-miSOD1 antibody did not bind to its epitope in most of the 41 postmortem spinal cord sections from non-neurological control (NNC) patients. In transgenic mice overexpressing disease-causing human SOD1G37R or SOD1G93A mutations, treatment with the α-miSOD1 antibody delayed the onset of motor symptoms, extended survival by up to 2 months, and reduced aggregation of misfolded SOD1 and motor neuron degeneration. These effects were obtained whether α-miSOD1 antibody treatment was administered by direct brain infusion or peripheral administration. These results support the further development of α-miSOD1 antibody as a candidate treatment for ALS involving misfolding of SOD1.

High-speed video gait analysis reveals early and characteristic locomotor phenotypes in mouse models of neurodegenerative movement disorders.

Neurodegenerative diseases of the central nervous system frequently affect the locomotor system resulting in impaired movement and gait. In this study we performed a whole-body high-speed video gait analysis in three different mouse lines of neurodegenerative movement disorders to investigate the motor phenotype. Based on precise computerized motion tracking of all relevant joints and the tail, a custom-developed algorithm generated individual and comprehensive locomotor profiles consisting of 164 spatial and temporal parameters. Gait changes observed in the three models corresponded closely to the classical clinical symptoms described in these disorders: Muscle atrophy due to motor neuron loss in SOD1 G93A transgenic mice led to gait characterized by changes in hind-limb movement and positioning. In contrast, locomotion in huntingtin N171-82Q mice modeling Huntington's disease with basal ganglia damage was defined by hyperkinetic limb movements and rigidity of the trunk. Harlequin mutant mice modeling cerebellar degeneration showed gait instability and extensive changes in limb positioning. Moreover, model specific gait parameters were identified and were shown to be more sensitive than conventional motor tests. Altogether, this technique provides new opportunities to decipher underlying disease mechanisms and test novel therapeutic approaches.

Complex interplay between brain function and structure during cerebral amyloidosis in APP transgenic mouse strains revealed by multi-parametric MRI comparison.

Alzheimer's disease is a fatal neurodegenerative disorder affecting the aging population. Neuroimaging methods, in particular magnetic resonance imaging (MRI), have helped reveal alterations in the brain structure, metabolism, and function of patients and in groups at risk of developing AD, yet the nature of these alterations is poorly understood. Neuroimaging in mice is attractive for investigating mechanisms underlying functional and structural changes associated with AD pathology. Several preclinical murine models of AD have been generated based on transgenic insertion of human mutated APP genes. Depending on the specific mutations, mouse strains express different aspects of amyloid pathology, e.g. intracellular amyloid-ß (Aß) aggregates, parenchymal plaques, or cerebral amyloid angiopathy. We have applied multi-parametric MRI in three transgenic mouse lines to compare changes in brain function with resting-state fMRI and structure with diffusion tensor imaging and high resolution anatomical imaging. E22ΔAß developing intracellular Aß aggregates did not present functional or structural alterations compared to their wild-type littermates. PSAPP mice displaying parenchymal amyloid plaques displayed mild functional changes within the supplementary and barrel field cortices, and increased isocortical volume relative to controls. Extensive reduction in functional connectivity in the sensory-motor cortices and within the default mode network, as well as local volume increase in the midbrain relative to wild-type have been observed in ArcAß mice bearing intracellular Aß aggregates as well as parenchymal and vascular amyloid deposits. Patterns of functional and structural changes appear to be strain-specific and not directly related to amyloid deposition.

Brain interstitial fluid glutamine homeostasis is controlled by blood-brain barrier SLC7A5/LAT1 amino acid transporter.

L-glutamine (Gln) is the most abundant amino acid in plasma and cerebrospinal fluid and a precursor for the main central nervous system excitatory (L-glutamate) and inhibitory (γ-aminobutyric acid (GABA)) neurotransmitters. Concentrations of Gln and 13 other brain interstitial fluid amino acids were measured in awake, freely moving mice by hippocampal microdialysis using an extrapolation to zero flow rate method. Interstitial fluid levels for all amino acids including Gln were ∼5-10 times lower than in cerebrospinal fluid. Although the large increase in plasma Gln by intraperitoneal (IP) injection of 15N2-labeled Gln (hGln) did not increase total interstitial fluid Gln, low levels of hGln were detected in microdialysis samples. Competitive inhibition of system A (SLC38A1&2; SNAT1&2) or system L (SLC7A5&8; LAT1&2) transporters in brain by perfusion with α-(methylamino)-isobutyric acid (MeAIB) or 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) respectively, was tested. The data showed a significantly greater increase in interstitial fluid Gln upon BCH than MeAIB treatment. Furthermore, brain BCH perfusion also strongly increased the influx of hGln into interstitial fluid following IP injection consistent with transstimulation of LAT1-mediated transendothelial transport. Taken together, the data support the independent homeostatic regulation of amino acids in interstitial fluid vs. cerebrospinal fluid and the role of the blood-brain barrier expressed SLC7A5/LAT1 as a key interstitial fluid gatekeeper.

Reduced ß-amyloid pathology in an APP transgenic mouse model of Alzheimer's disease lacking functional B and T cells.

INTRODUCTION: In Alzheimer's disease, accumulation and pathological aggregation of amyloid ß-peptide is accompanied by the induction of complex immune responses, which have been attributed both beneficial and detrimental properties. Such responses implicate various cell types of the innate and adaptive arm of the immunesystem, both inside the central nervous system, and in the periphery. To investigate the role of the adaptive immune system in brain ß-amyloidosis, PSAPP transgenic mice, an established mouse model of Alzheimer's disease, were crossbred with the recombination activating gene-2 knockout (Rag2 ko) mice lacking functional B and T cells. In a second experimental paradigm, aged PSAPP mice were reconstituted with bone marrow cells from either Rag2 ko or wildtype control mice. RESULTS: Analyses from both experimental approaches revealed reduced ß-amyloid pathology and decreased brain amyloid ß-peptide levels in PSAPP mice lacking functional adaptive immune cells. The decrease in brain ß-amyloid pathology was associated with enhanced microgliosis and increased phagocytosis of amyloid ß-peptide aggregates. CONCLUSION: The results of this study demonstrate an impact of the adaptive immunity on cerebral ß-amyloid pathology in vivo and suggest an influence on microglia-mediated amyloid ß-peptide clearance as a possible underlying mechanism.

Acute Effects of Muscarinic M1 Receptor Modulation on AßPP Metabolism and Amyloid-ß Levels in vivo: A Microdialysis Study.

Indirect modulation of cholinergic activity by cholinesterase inhibition is currently a widely established symptomatic treatment for Alzheimer's disease (AD). Selective activation of certain muscarinic receptor subtypes has emerged as an alternative cholinergic-based amyloid-lowering strategy for AD, as selective muscarinic M1 receptor agonists can reduce amyloid-ß (Aß) production by shifting endoproteolytic amyloid-ß protein precursor (AßPP) processing toward non-amyloidogenic pathways. In this study, we addressed the hypothesis that acute stimulation of muscarinic M1 receptors can inhibit Aß production in awake and freely moving AßPP transgenic mice. By combining intracerebral microdialysis with retrodialysis, we determined hippocampal Aß concentrations during simultaneous pharmacological modulation of brain M1 receptor function. Infusion with a M1 receptor agonist AF102B resulted in a rapid reduction of interstitial fluid (ISF) Aß levels while treatment with the M1 antagonist dicyclomine increased ISF Aß levels reaching significance within 120 minutes of treatment. The reduction in Aß levels was associated with PKCα and ERK activation resulting in increased levels of the α-secretase ADAM17 and a shift in AßPP processing toward the non-amyloidogenic processing pathway. In contrast, treatment with the M1 receptor antagonist dicyclomine caused a decrease in levels of phosphorylated ERK that was independent of PKCα, and led to an elevation of ß-secretase levels associated with increased amyloidogenic AßPP processing. The results of this study demonstrate rapid effects of in vivo M1 receptor modulation on the ISF pool of Aß and suggest that intracerebral microdialysis with retrodialysis is a useful technical approach for monitoring acute treatment effects of muscarinic receptor modulators on AßPP/Aß metabolism.

Amyloid-ß peptide-specific DARPins as a novel class of potential therapeutics for Alzheimer disease.

Passive immunization with anti-amyloid-ß peptide (Aß) antibodies is effective in animal models of Alzheimer disease. With the advent of efficient in vitro selection technologies, the novel class of designed ankyrin repeat proteins (DARPins) presents an attractive alternative to the immunoglobulin scaffold. DARPins are small and highly stable proteins with a compact modular architecture ideal for high affinity protein-protein interactions. In this report, we describe the selection, binding profile, and epitope analysis of Aß-specific DARPins. We further showed their ability to delay Aß aggregation and prevent Aß-mediated neurotoxicity in vitro. To demonstrate their therapeutic potential in vivo, mono- and trivalent Aß-specific DARPins (D23 and 3×D23) were infused intracerebroventricularly into the brains of 11-month-old Tg2576 mice over 4 weeks. Both D23 and 3×D23 treatments were shown to result in improved cognitive performance and reduced soluble Aß levels. These findings demonstrate the therapeutic potential of Aß-specific DARPins for the treatment of Alzheimer disease.

Quantifying uncertainty in Transcranial Magnetic Stimulation - A high resolution simulation study in ICBM space.

Transcranial Magnetic Stimulation offers enormous potential for noninvasive brain stimulation. While it is known that brain tissue significantly "reshapes" induced field and charge distributions, most modeling investigations to-date have focused on single-subject data with limited generality. Further, the effects of the significant uncertainties which exist in the simulation (i.e. brain conductivity distributions) and stimulation (e.g. coil positioning and orientations) setup have not been quantified. In this study, we construct a high-resolution anisotropic head model in standard ICBM space, which can be used as a population-representative standard for bioelectromagnetic simulations. Further, we employ Monte-Carlo simulations in order to quantify how uncertainties in conductivity values propagate all the way to induced field and currents, demonstrating significant, regionally dependent dispersions in values which are commonly assumed "ground truth". This framework can be leveraged in order to quantify the effect of any type of uncertainty in noninvasive brain stimulation and bears relevance in all applications of TMS, both investigative and therapeutic.

Transcranial magnetic stimulation in heterogeneous brain tissue: clinical impact on focality, reproducibility and true sham stimulation.

BACKGROUND: Transcranial magnetic stimulation (TMS) is an attractive research and possibly therapeutic tool for non-invasive central nervous system stimulation. However, relatively little is known about the direction, magnitude and distribution of induced electric field and current flows in tissue, and optimal setup characteristics as well as appropriate sham stimulation conditions remain largely undetermined, hampering reproducibility. METHODS: We reconstruct the conductive phenomena induced by TMS by implementing digitized coil geometry and realistic stimulator parameters and solving the electromagnetic problem over an MRI-based, realistic head model of 1mm resolution. Findings are validated by recording motor evoked potentials from the right abductor pollicis brevis muscle from healthy subjects stimulated in a stereotaxic framework. RESULTS: Several commonly used sham stimulation configurations elicit conductive patterns which achieve up to 40% of the strength of real stimulation. Also, variations in coil position of the order of a 7 degrees tilt, which are expected to occur in non-stereotaxic stimulation, can alter the stimulation intensity by up to 25%. CONCLUSIONS: In accordance with our findings, several clinical studies observe measurable effects during sham stimulation or no significant difference between sham and real stimulation, and the sensitivity of stimulation intensity to tiny coil rotations affords a partial explanation for the poor reproducibility and partial disagreements observed across clinical TMS studies. Knowledge of coil and stimulator specifications alone is hence not sufficient to control stimulation conditions, and a stereotaxic setup coupled with individually adjusted field solvers appear essential in performing reliable TMS studies.

A reconstruction of the conductive phenomena elicited by transcranial magnetic stimulation in heterogeneous brain tissue.

Transcranial magnetic stimulation is an attractive research and possibly therapeutic tool for non-invasive stimulation of brain tissue. However, relatively little is known about the direction, magnitude and distribution of induced fields and current flow in tissue, and optimal setup characteristics remain largely undetermined. Further, the profound influence of brain size and shape as well as of brain tissue irregularity on actual stimulation patterns is unclear. We model the conductive phenomena induced in brain tissue by TMS by solving the quasistatic problem over a realistic head model of 1mm resolution derived from anatomical MRI scans using a finite difference successive overrelaxation procedure. The magnetic field is calculated from digitized coil geometry and realistic stimulator parameters. Stimulation with a symmetrical primary electric field results in electric field and current density distributions which are highly asymmetrical both in magnitude and in direction (i.e. distributed, non-contiguous stimulation peaks, deviation of stimulated area from coil "hot spot", sudden jumps in stimulation intensity and non-zero current flow across tissue interfaces). Knowledge of coil and stimulator specifications alone is hence not sufficient to control stimulation conditions, and a stereotaxic setup coupled with an individually adjusted field solver appears essential in performing reliable TMS studies. Our results bear direct relevance to any application of TMS, both investigative and therapeutic.

Successful treatment of methicillin-resistant Staphylococcus aureus meningitis using linezolid without removal of intrathecal infusion pump. Case report.

Infection of an intrathecal pump system is a rare but serious complication and usually leads to the removal of the pump. The authors report the first case of methicillin-resistant Staphylococcus aureus (MRSA) meningitis in a patient with such a pump successfully treated with linezolid without the need for removal of the intrathecal pump. A 77-year-old woman with cervical myelopathy underwent implantation of an intrathecal pump system for baclofen administration. Two weeks after the procedure she developed meningitis caused by MRSA as isolated in cerebrospinal fluid (CSF) cultures, blood samples, and serum obtained from the pump pouch. Clinically she presented with meningism, somnolence, and signs of sepsis. When a combined intravenous antibiotic treatment regimen of vancomycin and rifampicin resulted in no clinical improvement, that regimen was discontinued and linezolid was administered intravenously as monotherapy. Within 3 days clinical and laboratory findings showed significant improvement. After 1 week of linezolid treatment, blood and CSF cultures were sterile. Intravenous treatment was administered for a total of 3 weeks, after which the patient was treated with oral linezolid for 3 months. During 18 months of follow-up, no new clinical or laboratory signs of infection were observed. These results confirm previous reports of the efficacy of linezolid for the treatment of severe infections of the central nervous system caused by multidrug-resistant Gram-positive bacteria, especially postneurosurgical infections.

Polymorphisms in the serotonin receptor gene HTR2A are associated with quantitative traits in panic disorder.

Anxiety disorders and specifically panic disorder (PD) are caused by complex interactions of environmental and genetic factors. The latter comprise many different genes, from which those involved in serotonergic neurotransmission have received particular attention. Here we report the results from an association candidate-gene approach, where we analyzed 15 single nucleotide polymorphisms (SNPs) within the gene coding for the serotonin-receptor 2A (HTR2A) in patients suffering from PD and a control sample. We found that the SNP rs2296972 shows an association between the number of T-alleles and severity of symptoms in PD. By performing tests according to the Fisher product method (FPM), an association between HTR2A and the personality trait reward dependence could be shown. Most pronounced effects were observable for the SNPs rs2770304, rs6313, and rs6311. Furthermore, the polymorphisms rs3742278, rs2296972, and rs2770292 form a haplotype, which may be associated with higher susceptibility for PD. These results further underline a possible important role of genetic variations within the system controlling serotonergic neurotransmission for the development and course of disease in PD.

Temazepam triggers the release of vasopressin into the rat hypothalamic paraventricular nucleus: novel insight into benzodiazepine action on hypothalamic-pituitary-adrenocortical system activity during stress.

We investigated the influence of a representative classical benzodiazepine on the regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis activity both under basal conditions and stress. Adult male Wistar rats were intravenously administered with temazepam (0.5, 1, and 3 mg/kg body weight) and plasma concentrations of corticotropin (ACTH) and vasopressin (AVP) were measured in blood samples collected via chronically implanted jugular venous catheters. Simultaneously, the release of AVP within the hypothalamic paraventricular nucleus (PVN) was monitored via microdialysis. Plasma AVP levels remained unaffected by the different treatment conditions. Temazepam blunted the stressor exposure-induced secretion of ACTH in a dose-dependent manner. Concurrently, and also in a dose-dependent manner temazepam enhanced the intra-PVN release of AVP, known to originate from magnocellular neurons of the hypothalamic neurohypophyseal system. Furthermore, temazepam did not affect the in vitro secretion of ACTH from the adenohypophyseal cells. Taken together, the results of this study suggest that temazepam modulates the central nervous regulation of the HPA axis by altering intra-PVN AVP release. An increasingly released AVP of magnocellular origin seems to provide a negative tonus on ACTH secretion most probably via inhibiting the release of ACTH secretagogues from the median eminence into hypophyseal portal blood.

Differences in serotonergic neurotransmission between rats displaying high or low anxiety/depression-like behaviour: effects of chronic paroxetine treatment.

Disturbances in serotonergic neurotransmission have been suggested to be closely interlinked with hyperactivity of the hypothalamic-pituitary-adrenocortical (HPA) system, and are likely to be involved in the pathophysiology of anxiety disorders and major depression. We therefore investigated markers of serotonergic transmission and their modulation by chronic paroxetine in rats selectively bred for high (HAB) or low (LAB) anxiety-related behaviour, both under basal conditions and in response to emotional stress. Hippocampal serotonin 1 A (5-HT1A) receptor mRNA expression was reduced in HAB rats, whereas 5-HT concentrations in hippocampal microdialysates did not differ between HAB and LAB rats under basal conditions. In the hippocampus, overall expression of serotonin transporter binding sites was increased in HAB compared with LAB rats. Exposure to emotional stress failed to increase intrahippocampal 5-HT release in HAB rats whereas LAB rats displayed a physiological, albeit small rise. Chronic paroxetine treatment markedly increased the stress-induced rise in hippocampal 5-HT in HAB, but not LAB rats. This effect may be (at least in part) related to a greater down-regulation of hippocampal serotonin transporter binding sites by paroxetine in HABs compared with LABs, while 5-HT1A receptor expression remained unaffected in this brain area. The findings indicate reduced hippocampal serotonergic transmission in HAB rats as compared with LAB rats, which is evident both at the presynaptic (5-HT release) and the postsynaptic (5-HT1A receptor) level. Chronic paroxetine enhanced the presynaptic responsivity in HAB rats, but not LAB rats, pointing to a preferential efficacy of paroxetine in rats with enhanced anxiety/depression-related behaviour.

Reduction of hypothalamic vasopressinergic hyperdrive contributes to clinically relevant behavioral and neuroendocrine effects of chronic paroxetine treatment in a psychopathological rat model.

The neuroendocrine and behavioral effects of chronic paroxetine treatment were investigated in two rat lines selectively bred for high anxiety-related behavior (HAB) or low anxiety-related behavior (LAB) emotionality. In addition to a characteristic behavioral phenotype with markedly passive stress-coping strategies, HAB rats show a hypothalamic vasopressinergic hyperdrive that is causally related to hypothalamic-pituitary-adrenocortical dysregulation as demonstrated in the combined dexamethasone (DEX)/corticotropin-releasing hormone (CRH) test. A total of 8 weeks of chronic paroxetine treatment induced a more active coping strategy in the forced swim test in HAB rats only. In contrast, paroxetine-treated LAB rats did not change their swimming behavior. To investigate the neuroendocrine alterations linked to these behavioral changes, a combined DEX/CRH test was performed. In HAB rats, the paroxetine-induced behavioral changes towards more active coping strategies were accompanied by a normalization of the CRH-stimulated increase in corticotropin (ACTH) and corticosterone secretion. Concomitantly, the hypothalamic vasopressinergic hyperdrive was found to be reduced in HAB but not LAB rats, as indicated by a decrease in vasopressin mRNA expression, whereas vasopressin 1a receptor binding was unaffected. These findings provide the first evidence that the vasopressinergic system is likely to be critically involved in the behavioral and neuroendocrine effects of antidepressant drugs. This novel mechanism of action of paroxetine on vasopressin gene regulation renders vasopressinergic neuronal circuits a promising target for the development of more causal antidepressant treatment strategies.

Chronic psychosocial stress and concomitant repetitive transcranial magnetic stimulation: effects on stress hormone levels and adult hippocampal neurogenesis.

BACKGROUND: Repetitive transcranial magnetic stimulation is increasingly used as a therapeutic tool in psychiatry and has been demonstrated to attenuate the activity of the stress hormone system. Stress-induced structural remodeling in the adult hippocampus may provide a cellular basis for understanding the impairment of neural plasticity in depressive illness. Accordingly, reversal of structural remodeling might be a desirable goal for antidepressant therapy. The present study investigated the effect of chronic psychosocial stress and concomitant repetitive transcranial magnetic stimulation treatment on stress hormone regulation and hippocampal neurogenesis. METHODS: Adult male rats were submitted to daily psychosocial stress and repetitive transcranial magnetic stimulation (20 Hz) for 18 days. Cell proliferation in the dentate gyrus was quantified by using BrdU immunohistochemistry, and both the proliferation rate of progenitors and the survival rate of BrdU-labeled cells were evaluated. To characterize the activity of the hypothalamic-pituitary-adrenocortical system, plasma corticotropin and corticosterone concentrations were measured. RESULTS: Chronic psychosocial stress resulted in a significant increase of stress hormone levels and potently suppressed the proliferation rate and survival of the newly generated hippocampal granule cells. Concomitant repetitive transcranial magnetic stimulation treatment normalized the stress-induced elevation of stress hormones; however, despite the normalized activity of the hypothalamic-pituitary-adrenocortical system, the decrement of hippocampal cell proliferation was only mildly attenuated by repetitive transcranial magnetic stimulation, while the survival rate of BrdU-labeled cells was further suppressed by the treatment. CONCLUSIONS: These results support the notion that attenuation of the hypothalamic-pituitary-adrenocortical system is an important mechanism underlying the clinically observed antidepressant effect of repetitive transcranial magnetic stimulation, whereas this experimental design did not reveal beneficial effects of repetitive transcranial magnetic stimulation on adult hippocampal neurogenesis.

Vasopressin mediates the response of the combined dexamethasone/CRH test in hyper-anxious rats: implications for pathogenesis of affective disorders.

To investigate the neuroendocrine alterations linked to inborn emotionality in two Wistar rat lines selectively bred for either high (HAB) or low (LAB) anxiety-related behavior, we administered the combined dexamethasone (DEX)/corticotropin-releasing hormone (CRH) test. DEX (12:00 M. (noon); 30 microg/kg) resulted in a significantly less efficient suppression of the diurnal increase in the circulating corticotropin (ACTH) levels in the male HAB rats than in the male LAB rats. In addition, plasma ACTH and corticosterone responses to subsequent CRH (7:30 P.M.; 50 ng/kg) were significantly higher in male HAB rats. The rise in ACTH after CRH in the DEX-pretreated male HAB rats points toward an enhanced activity and involvement of endogenous vasopressin synthesized in the hypothalamic paraventricular nucleus (PVN) and acting at pituitary corticotrope cells. We tested this hypothesis by in situ hybridization and in vivo microdialysis, and found an increase in both basal synthesis and release of vasopressin within the PVN of the male HAB rats. As expected, pretreatment with a selective vasopressin type 1 receptor antagonist abolished the CRH-stimulated increase in ACTH secretion in the DEX-pretreated male HAB rats. The results indicate that vasopressin-mediated effects are critically involved in the profound disturbance of the hypothalamic-pituitary-adrenocortical system in male HAB rats, thus revealing striking parallels to the neuroendocrine situation in human depression.