Early diagnosis and treatment of Alzheimer's disease by targeting toxic soluble Aß oligomers.

Transient soluble oligomers of amyloid-ß (Aß) are toxic and accumulate early prior to insoluble plaque formation and cognitive impairment in Alzheimer's disease (AD). Synthetic cyclic D,L-α-peptides (e.g., 1) self-assemble into cross ß-sheet nanotubes, react with early Aß species (1-3 mers), and inhibit Aß aggregation and toxicity in stoichiometric concentrations, in vitro. Employing a semicarbazide as an aza-glycine residue with an extra hydrogen-bond donor to tune nanotube assembly and amyloid engagement, [azaGly6]-1 inhibited Aß aggregation and toxicity at substoichiometric concentrations. High-resolution NMR studies revealed dynamic interactions between [azaGly6]-1 and Aß42 residues F19 and F20, which are pivotal for early dimerization and aggregation. In an AD mouse model, brain positron emission tomography (PET) imaging using stable 64Cu-labeled (aza)peptide tracers gave unprecedented early amyloid detection in 44-d presymptomatic animals. No tracer accumulation was detected in the cortex and hippocampus of 44-d-old 5xFAD mice; instead, intense PET signal was observed in the thalamus, from where Aß oligomers may spread to other brain parts with disease progression. Compared with standard 11C-labeled Pittsburgh compound-B (11C-PIB), which binds specifically fibrillar Aß plaques, 64Cu-labeled (aza)peptide gave superior contrast and uptake in young mouse brain correlating with Aß oligomer levels. Effectively crossing the blood-brain barrier (BBB), peptide 1 and [azaGly6]-1 reduced Aß oligomer levels, prolonged lifespan of AD transgenic Caenorhabditis elegans, and abated memory and behavioral deficits in nematode and murine AD models. Cyclic (aza)peptides offer novel promise for early AD diagnosis and therapy.

Neuronal deletion of Gtf2i results in developmental microglial alterations in a mouse model related to Williams syndrome.

Williams syndrome (WS) is a genetic neurodevelopmental disorder caused by a heterozygous microdeletion, characterized by hypersociability and unique neurocognitive abnormalities. Of the deleted genes, GTF2I has been linked to hypersociability in WS. We have recently shown that Gtf2i deletion from forebrain excitatory neurons, referred to as Gtf2i conditional knockout (cKO) mice leads to multi-faceted myelination deficits associated with the social behaviors affected in WS. These deficits were potentially mediated also by microglia, as they present a close relationship with oligodendrocytes. To study the impact of altered myelination, we characterized these mice in terms of microglia over the course of development. In postnatal day 30 (P30) Gtf2i cKO mice, cortical microglia displayed a more ramified state, as compared with wild type (controls). However, postnatal day 4 (P4) microglia exhibited high proliferation rates and an elevated activation state, demonstrating altered properties related to activation and inflammation in Gtf2i cKO mice compared with control. Intriguingly, P4 Gtf2i cKO-derived microglial cells exhibited significantly elevated myelin phagocytosis in vitro compared to control mice. Lastly, systemic injection of clemastine to P4 Gtf2i cKO and control mice until P30, led to a significant interaction between genotypes and treatments on the expression levels of the phagocytic marker CD68, and a significant reduction of the macrophage/microglial marker Iba1 transcript levels in the cortex of the Gtf2i cKO treated mice. Our data thus implicate microglia as important players in WS, and that early postnatal manipulation of microglia might be beneficial in treating inflammatory and myelin-related pathologies.

Extracellular DAMPs in Plants and Mammals: Immunity, Tissue Damage and Repair.

Innate immune receptors, well known mediators of response to non-self-molecules and inflammation, also act as mediators of immunity triggered by 'damage-associated molecular patterns' (DAMPs). Pathogen-associated molecular patterns (PAMPs) cause inflammation in mammals and a rapid immune response in plants, while DAMPs trigger more complex responses, including immunity, tissue maintenance and repair. DAMPs, their receptors and downstream transduction mechanisms are often conserved within a kingdom or, due to convergent evolution, are similar across the kingdoms of life. Herein, we describe the dynamics and functionality of specific extracellular DAMP classes and their receptors in immunity, inflammation and repair of tissue damage in plants and mammals.

Restoring microglial and astroglial homeostasis using DNA immunization in a Down Syndrome mouse model.

Down Syndrome (DS), the most common cause of genetic intellectual disability, is characterized by over-expression of the APP and DYRK1A genes, located on the triplicated chromosome 21. This chromosomal abnormality leads to a cognitive decline mediated by Amyloid-ß (Aß) overproduction and tau hyper-phosphorylation as early as the age of 40. In this study, we used the Ts65Dn mouse model of DS to evaluate the beneficial effect of a DNA vaccination against the Aß1-11 fragment, in ameliorating Aß-related neuropathology and rescue of cognitive and behavioral abilities. Anti-Aß1-11 vaccination induced antibody production and facilitated clearance of soluble oligomers and small extracellular inclusions of Aß from the hippocampus and cortex of Ts65Dn mice. This was correlated with reduced neurodegeneration and restoration of the homeostatic phenotype of microglial and astroglial cells. Vaccinated Ts65Dn mice performed better in spatial-learning tasks, exhibited reduced motor hyperactivity typical for this strain, and restored short-term memory abilities. Our findings support the hypothesis that DS individuals may benefit from active immunotherapy against Aß from a young age by slowing the progression of dementia.

Dopaminergic Modulation of Synaptic Integration and Firing Patterns in the Rat Entopeduncular Nucleus.

Dopamine is known to differentially modulate the impact of cortical input to the striatum between the direct and indirect pathways of the basal ganglia (BG). However, the role of extrastriatal dopamine receptors (DRs) in BG information processing is less clear. To investigate the role of extrastriatal DRs, we studied their distribution and function in one of the output nuclei of the BG of the rodent, the entopeduncular nucleus (EP). qRT-PCR indicated that all DR subtypes were expressed by EP neurons, suggesting that both D1-like receptors (D1LRs) and D2-like receptors (D2LRs) were likely to affect information processing in the EP. Whole-cell recordings revealed that striatal inputs to the EP were potentiated by D1LRs whereas pallidal inputs to the EP were depressed by D2LRs. Changes to the paired-pulse ratio of inputs to the EP suggested that dopaminergic modulation of striatal inputs is mediated by postsynaptic receptors, and that of globus pallidus-evoked inputs is mediated by presynaptic receptors. We show that these changes in synaptic efficacy changed the information content of EP neuron firing. Overall, the findings suggest that the dopaminergic system affects the passage of feedforward information through the BG by modulating input divergence in the striatum and output convergence in the EP.SIGNIFICANCE STATEMENT The entopeduncular nucleus (EP), one of the basal ganglia (BG) output nuclei, is an important station in information processing in BG. However, it remains unclear how EP neurons encode information and how dopamine affects this process. This contrasts with the well established role of dopamine in the striatum, which is known to redistribute cortical input between the direct and indirect pathways. Here we show that, in symmetry with the striatum, dopamine controls the rebalancing of information flow between the two pathways in the EP. Specifically, we demonstrate that dopamine regulates EP activity by differentially modulating striatal and pallidal GABAergic inputs. These results call for a reassessment of current perspectives on BG information processing by highlighting the functional role of extrastriatal dopamine receptors.

Unbiased classification of spatial strategies in the Barnes maze.

MOTIVATION: Spatial learning is one of the most widely studied cognitive domains in neuroscience. The Morris water maze and the Barnes maze are the most commonly used techniques to assess spatial learning and memory in rodents. Despite the fact that these tasks are well-validated paradigms for testing spatial learning abilities, manual categorization of performance into behavioral strategies is subject to individual interpretation, and thus to bias. We have previously described an unbiased machine-learning algorithm to classify spatial strategies in the Morris water maze. RESULTS: Here, we offer a support vector machine-based, automated, Barnes-maze unbiased strategy (BUNS) classification algorithm, as well as a cognitive score scale that can be used for memory acquisition, reversal training and probe trials. The BUNS algorithm can greatly benefit Barnes maze users as it provides a standardized method of strategy classification and cognitive scoring scale, which cannot be derived from typical Barnes maze data analysis. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://okunlab.wix.com/okunlab as a MATLAB application. CONTACT: eitan.okun@biu.ac.ilSupplementary information: Supplementary data are available at Bioinformatics online.

Toll-like receptor 3 deficiency decreases epileptogenesis in a pilocarpine model of SE-induced epilepsy in mice.

OBJECTIVE: Epilepsy affects 60 million people worldwide. Despite the development of antiepileptic drugs, up to 35% of patients are drug refractory with uncontrollable seizures. Toll-like receptors (TLRs) are central components of the nonspecific innate inflammatory response. Because TLR3 was recently implicated in neuronal plasticity, we hypothesized that it may contribute to the development of epilepsy after status epilepticus (SE). METHODS: To test the involvement of TLR3 in epileptogenesis, we used the pilocarpine model for SE in TLR3-deficient mice and their respective wild-type controls. In this model, a single SE event leads to spontaneous recurrent seizures (SRS). Two weeks after SE, mice were implanted with wireless electroencephalography (EEG) transmitters for up to 1 month. The impact of TLR3 deficiency on SE was assessed using separate cohorts of mice regarding EEG activity, seizure progression, hippocampal microglial distribution, and expression of the proinflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)ß. RESULTS: Our data indicate that TLR3 deficiency reduced SRS, microglial activation, and the levels of the proinflammatory cytokines TNFα and IFNß, and increased survival following SE. SIGNIFICANCE: This study reveals novel insights into the pathophysiology of epilepsy and the contribution of TLR3 to disease progression. Our results identify the TLR3 pathway as a potential future therapeutic target in SE.

Unraveling cognitive traits using the Morris water maze unbiased strategy classification (MUST-C) algorithm.

The assessment of spatial cognitive learning in rodents is a central approach in neuroscience, as it enables one to assess and quantify the effects of treatments and genetic manipulations from a broad perspective. Although the Morris water maze (MWM) is a well-validated paradigm for testing spatial learning abilities, manual categorization of performance in the MWM into behavioral strategies is subject to individual interpretation, and thus to biases. Here we offer a support vector machine (SVM) - based, automated, MWM unbiased strategy classification (MUST-C) algorithm, as well as a cognitive score scale. This model was examined and validated by analyzing data obtained from five MWM experiments with changing platform sizes, revealing a limitation in the spatial capacity of the hippocampus. We have further employed this algorithm to extract novel mechanistic insights on the impact of members of the Toll-like receptor pathway on cognitive spatial learning and memory. The MUST-C algorithm can greatly benefit MWM users as it provides a standardized method of strategy classification as well as a cognitive scoring scale, which cannot be derived from typical analysis of MWM data.

Postnatal TLR2 activation impairs learning and memory in adulthood.

Neuroinflammation in the central nervous system is detrimental for learning and memory, as evident form epidemiological studies linking developmental defects and maternal exposure to harmful pathogens. Postnatal infections can also induce neuroinflammatory responses with long-term consequences. These inflammatory responses can lead to motor deficits and/or behavioral disabilities. Toll like receptors (TLRs) are a family of innate immune receptors best known as sensors of microbial-associated molecular patterns, and are the first responders to infection. TLR2 forms heterodimers with either TLR1 or TLR6, is activated in response to gram-positive bacterial infections, and is expressed in the brain during embryonic development. We hypothesized that early postnatal TLR2-mediated neuroinflammation would adversely affect cognitive behavior in the adult. Our data indicate that postnatal TLR2 activation affects learning and memory in adult mice in a heterodimer-dependent manner. TLR2/6 activation improved motor function and fear learning, while TLR2/1 activation impaired spatial learning and enhanced fear learning. Moreover, developmental TLR2 deficiency significantly impairs spatial learning and enhances fear learning, stressing the involvement of the TLR2 pathway in learning and memory. Analysis of the transcriptional effects of TLR2 activation reveals both common and unique transcriptional programs following heterodimer-specific TLR2 activation. These results imply that adult cognitive behavior could be influenced in part, by activation or alterations in the TLR2 pathway at birth.

Evidence that collaboration between HIF-1α and Notch-1 promotes neuronal cell death in ischemic stroke.

Recent findings suggest that Notch-1 signaling contributes to neuronal death in ischemic stroke, but the underlying mechanisms are unknown. Hypoxia inducible factor-1α (HIF-1α), a global regulator of cellular responses to hypoxia, can interact with Notch and modulate its signaling during hypoxic stress. Here we show that Notch signaling interacts with the HIF-1α pathway in the process of ischemic neuronal death. We found that a chemical inhibitor of the Notch-activating enzyme, γ-secretase, and a HIF-1α inhibitor, protect cultured cortical neurons against ischemic stress, and combined inhibition of Notch-1 and HIF-1α further decreased neuronal death. HIF-1α and Notch intracellular domain (NICD) are co-expressed in the neuronal nucleus, and co-immunoprecipitated in cultured neurons and in brain tissue from mice subjected to focal ischemic stroke. Overexpression of NICD and HIF-1α in cultured human neural cells enhanced cell death under ischemia-like conditions, and a HIF-1α inhibitor rescued the cells. RNA interference-mediated depletion of endogenous NICD and HIF-1α also decreased cell death under ischemia-like conditions. Finally, mice treated with inhibitors of γ-secretase and HIF-1α exhibited improved outcome after focal ischemic stroke, with combined treatment being superior to individual treatments. Additional findings suggest that the NICD and HIF-1α collaborate to engage pro-inflammatory and apoptotic signaling pathways in stroke.

Toll-like receptors 2 and 4 modulate autonomic control of heart rate and energy metabolism.

Toll-like receptors (TLR) are innate immune receptors typically activated by microbial-associated molecular patterns (MAMPs) during infection or damage-associated molecular patterns (DAMPs) as a result of tissue injury. Recent findings suggest that TLR2 and TLR4 signaling play important roles in developmental and adult neuroplasticity, and in learning and memory. In addition, activation of TLR2 and TLR4 worsens ischemic injury to the heart and brain in animal models of myocardial infarction and stroke. TLR activation is also implicated in thermoregulation and fever in response to infection. However, it is not known whether TLRs participate in the regulation of the sympathetic and/or parasympathetic components of the autonomic nervous system (ANS). Here we provide evidence that TLR2 and TLR4 influence autonomic regulation of heart rate (HR) body temperature and energy metabolism in mice. We show that mice lacking TLR2 or TLR4 exhibit reduced basal HR, which results from an increase of parasympathetic tone. In addition, thermoregulatory responses to stress are altered in TLR2-/- and TLR4-/- mice, and brown fat-dependent thermoregulation is altered in TLR4-/- mice. Moreover, TLR2-/- and TLR4-/- mice consume less food and exhibit a greater mass compared to wild type mice. Collectively, our findings suggest important roles for TLR2 and TLR4 in the ANS regulation of cardiovascular function, thermoregulation, and energy metabolism.

Electroconvulsive shock ameliorates disease processes and extends survival in huntingtin mutant mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by expanded polyglutamine repeats in the huntingtin (Htt) protein. Mutant Htt may damage and kill striatal neurons by a mechanism involving reduced production of brain-derived neurotrophic factor (BDNF) and increased oxidative and metabolic stress. Because electroconvulsive shock (ECS) can stimulate the production of BDNF and protect neurons against stress, we determined whether ECS treatment would modify the disease process and provide a therapeutic benefit in a mouse model of HD. ECS (50 mA for 0.2 s) or sham treatment was administered once weekly to male N171-82Q Htt mutant mice beginning at 2 months of age. Endpoints measured included motor function, striatal and cortical pathology, and levels of protein chaperones and BDNF. ECS treatment delayed the onset of motor symptoms and body weight loss and extended the survival of HD mice. Striatal neurodegeneration was attenuated and levels of protein chaperones (Hsp70 and Hsp40) and BDNF were elevated in striatal neurons of ECS-treated compared with sham-treated HD mice. Our findings demonstrate that ECS can increase the resistance of neurons to mutant Htt resulting in improved functional outcome and extended survival. The potential of ECS as an intervention in subjects that inherit the mutant Htt gene merits further consideration.

Gamma secretase-mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke.

Mice transgenic for antisense Notch and normal mice treated with inhibitors of the Notch-activating enzyme gamma-secretase showed reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Notch endangers neurons by modulating pathways that increase their vulnerability to apoptosis, and by activating microglial cells and stimulating the infiltration of proinflammatory leukocytes. These findings suggest that Notch signaling may be a therapeutic target for treatment of stroke and related neurodegenerative conditions.

Toll-like receptor 3 inhibits memory retention and constrains adult hippocampal neurogenesis.

Toll-like receptors (TLRs) are innate immune receptors that have recently emerged as regulators of neuronal survival and developmental neuroplasticity. Adult TLR3-deficient mice exhibited enhanced hippocampus-dependent working memory in the Morris water maze, novel object recognition, and contextual fear-conditioning tasks. In contrast, TLR3-deficient mice demonstrated impaired amygdala-related behavior and anxiety in the cued fear-conditioning, open field, and elevated plus maze tasks. Further, TLR3-deficient mice exhibited increased hippocampal CA1 and dentate gyrus volumes, increased hippocampal neurogenesis, and elevated levels of the AMPA receptor subunit GluR1 in the CA1 region of the hippocampus. In addition, levels of activated forms of the kinase ERK and the transcription factor CREB were elevated in the hippocampus of TLR3-deficient mice, suggesting that constitutive TLR3 signaling negatively regulates pathways known to play important roles in hippocampal plasticity. Direct activation of TLR3 by intracerebroventricular infusion of a TLR3 ligand impaired working memory, but not reference memory. Our findings reveal previously undescribed roles for TLR3 as a suppressor of hippocampal cellular plasticity and memory retention.

Neural correlates of subjective cognitive decline in adults at high risk for Alzheimer's disease.

Introduction: Recently, interest has emerged in subjective cognitive decline (SCD) as a potential precursor to Alzheimer's disease (AD) dementia. Whether individuals with SCD harbor brain alterations in midlife, when AD-related pathology begins, is yet to be elucidated. Furthermore, the role of apolipoprotein ε4 (APOE ε4) allele, a robust AD risk factor, in the relationship between SCD and brain alterations is unknown. We examined whether APOE genotype modulates the association of SCD with brain measures in individuals at high AD risk. Methods: Middle-aged adults with parental history of AD dementia underwent magnetic resonance imaging (MRI) and the Memory Functioning Questionnaire. Regression analysis tested the extent to which SCD was associated with activation during an functional MRI (fMRI) working-memory task, and white-matter microstructure. APOE ε4 genotype was tested as a moderator. Results: Among APOE ε4 carriers, but not among non-carriers, SCD was associated with higher activation in the anterior cingulate (p = 0.003), inferior, middle, and superior frontal cortices (p = 0.041, p = 0.048, p = 0.037, respectively); and with lower fractional anisotropy in the uncinate fasciculus (p = 0.002), adjusting for age, sex, and education. Conclusion: In middle aged, cognitively normal individuals at high AD risk, higher SCD was associated with greater brain alterations possibly reflecting incipient AD pathology. When accompanied by a family history of AD and an APOE ε4 allele, SCD may have important clinical value, allowing a window for early intervention and for participants' stratification in AD prevention clinical trials.

HCAR1-Mediated L-Lactate Signaling Suppresses Microglial Phagocytosis.

Microglia, the primary brain-resident immune cells, protect the brain from various harmful pathogens, insulting and maintaining its homeostasis by phagocytosing extracellular particles. How microglia are metabolically regulated by their microenvironment remains largely elusive. Here, we investigated how extracellular lactate, which is abundant in the brain and dynamically changes in pathological states, affects microglial phagocytotic ability. We show that L-lactate reduces microglia phagocytic capacity in a Hydroxycarboxylic Acid Receptor 1 but not Monocarboxylate transporter 1-dependent manner. Our findings point to a potential role for extracellular lactate in suppressing the phagocytic activity of microglial cells in homeostasis and inflammatory conditions.

Physiology and pharmacology of amyloid precursor protein.

Amyloid precursor protein (APP) is an evolutionarily conserved transmembrane protein and a well-characterized precursor protein of amyloid-beta (Aß) peptides, which accumulate in the brains of individuals with Alzheimer's disease (AD)-related pathologies. Aß has been extensively investigated since the amyloid hypothesis in AD was proposed. Besides Aß, previous studies on APP and its proteolytic cleavage products have suggested their diverse pathological and physiological functions. However, their roles still have not been thoroughly understood. In this review, we extensively discuss the evolutionarily-conserved biology of APP, including its structure and processing pathway, as well as recent findings on the physiological roles of APP and its fragments in the central nervous system and peripheral nervous system. We have also elaborated upon the current status of APP-targeted therapeutic approaches for AD treatment by discussing inhibitors of several proteases participating in APP processing, including α-, ß-, and γ-secretases. Finally, we have highlighted the future perspectives pertaining to further research and the potential clinical role of APP.

Sleeve Gastrectomy Reduces Glycemia but Does Not Affect Cognitive Impairment in Lean 5xFAD Mice.

Obesity and hyperglycemia are risk factors for cognitive decline and for the development of Alzheimer's Disease (AD). Bariatric surgery is an effective treatment for obesity that was shown to improve cognitive decline in obese patients. Bariatric surgery was shown to exert weight loss independent effects on metabolic diseases such as type 2 diabetes. We tested whether sleeve gastrectomy (SG), a common bariatric surgery, can affect the cognitive impairment in lean, normoglycemic female 5xFAD mice, a genetic model for AD. 5xFAD mice and wild-type (WT) littermates underwent SG or sham surgery at the age of 5 months and were tested for metabolic, behavioral, and molecular phenotypes 90 days later. SG led to a reduction in blood glucose levels and total plasma cholesterol levels in 5xFAD mice without inducing weight loss. However, the surgery did not affect the outcomes of long-term spatial memory tests in these mice. Analysis of ß-Amyloid plaques corroborated the behavioral studies in showing no effect of surgery on the molecular phenotype of 5xFAD mice. In conclusion, SG leads to an improved metabolic profile in lean female 5xFAD mice without inducing weight loss but does not affect the brain pathology or behavioral phenotype. Our results suggest that the positive effects of bariatric surgery on cognitive decline in obese patients are likely attributed to weight loss and improvement in obesity sequelae, and not to weight loss independent effects of surgery.