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.

Induction of an effective anti-Amyloid-ß humoral response in aged mice.

Aging-related decline in immune functions, termed immunosenescence, is a primary cause of reduced protective responses to vaccines in the elderly, due to impaired induction of cellular and humoral responses to new antigens (Ag), especially if the response is T cell dependent. The result is a more severe morbidity following infections, more prolonged and frequent hospitalization, and a higher mortality rate than in the general population. Therefore, there is an increasing need to develop vaccination strategies that overcome immunosenescence, especially for aging-related diseases such as Alzheimer's disease (AD). Here we report a new vaccination strategy harnessing memory-based immunity, which is less affected by aging. We found that aged C57BL/6 and 5xFAD mice exhibit a dramatic reduction in anti-Amyloid-ß (Aß) antibody (Ab) production. We aimed to reverse this process by inducing memory response at a young age. To this end, young mice were primed with the vaccine carrier Hepatitis B surface antigen (HBsAg). At an advanced age, these mice were immunized with an Aß1-11 fused to HBsAg. This vaccination scheme elicited a markedly higher Aß-specific antibody titer than vaccinating aged unprimed mice with the same construct. Importantly, this vaccine strategy more efficiently reduced cerebral Aß levels and altered microglial phenotype. Overall, we provide evidence that priming with an exogenous Ag carrier can overcome impaired humoral responses to self-antigens in the elderly, paving the route for a potent immunotherapy to AD.

Maternal antibodies facilitate Amyloid-ß clearance by activating Fc-receptor-Syk-mediated phagocytosis.

Maternal antibodies (MAbs) protect against infections in immunologically-immature neonates. Maternally transferred immunity may also be harnessed to target diseases associated with endogenous protein misfolding and aggregation, such as Alzheimer's disease (AD) and AD-pathology in Down syndrome (DS). While familial early-onset AD (fEOAD) is associated with autosomal dominant mutations in the APP, PSEN1,2 genes, promoting cerebral Amyloid-ß (Aß) deposition, DS features a life-long overexpression of the APP and DYRK1A genes, leading to a cognitive decline mediated by Aß overproduction and tau hyperphosphorylation. Although no prenatal screening for fEOAD-related mutations is in clinical practice, DS can be diagnosed in utero. We hypothesized that anti-Aß MAbs might promote the removal of early Aß accumulation in the central nervous system of human APP-expressing mice. To this end, a DNA-vaccine expressing Aß1-11 was delivered to wild-type female mice, followed by mating with 5xFAD males, which exhibit early Aß plaque formation. MAbs reduce the offspring's cortical Aß levels 4 months after antibodies were undetectable, along with alleviating short-term memory deficits. MAbs elicit a long-term shift in microglial phenotype in a mechanism involving activation of the FcγR1/Syk/Cofilin pathway. These data suggest that maternal immunization can alleviate cognitive decline mediated by early Aß deposition, as occurs in EOAD and DS.

Proteinoid Nanocapsules as Drug Delivery System for Improving Antipsychotic Activity of Risperidone.

Risperidone (RSP) is an atypical antipsychotic drug widely used to treat schizophrenia and bipolar disorder. Nanoparticles (NPs) are being developed as in vivo targeted drug delivery systems, which cross the blood-brain barrier and improve pharmacokinetics and drug effectiveness. Here, biodegradable proteinoids were synthesized by thermal step-growth polymerization from the amino acids l-glutamic acid, l-phenylalanine and l-histidine and poly (l-lactic acid). Proteinoid NPs containing RSP were then formed by self-assembly, overcoming the insolubility of the drug in water, followed by PEGylation (poly ethylene glycol (PEG) conjugation to increase the stability of the NPs in the aqueous continuous phase. These NPs are biodegradable owing to their peptide and ester moieties. They were characterized in terms of diameter, size distribution, drug loading, and long-term storage. Behavioral studies on mice found enhanced antipsychotic activity compared to free RSP.

A modified Barnes maze for an accurate assessment of spatial learning in mice.

BACKGROUND: The Morris water maze (MWM) and the Barnes maze (BM) are among the most widely-used paradigms for assessing spatial learning in rodents, with specific advantages and disadvantages for each apparatus. Compared with the intense water-related stress exerted during the MWM, the BM exhibits a milder light-induced stress, while suffering from biasing animals towards non-spatial strategies such as serial search, a heuristic non-spatial search strategy. To overcome this problem, we have developed a modified Barnes maze (MBM) apparatus that recapitulates natural environments more accurately without inducing undesirable exploration strategy bias. NEW METHOD: Apparatus. A circular 122 cm-wide table with 40 randomly placed holes. One target hole is leading to an escape chamber. Task. Three target locations were examined, varying in their distance from the center. C57BL6/j male mice were given three trials per day to find the target. Following acquisition, a probe test was performed by removing the escape chamber. RESULTS: Spatial-encoding-depended reduction in latency to reach the target was observed, along with improvement in path efficiency with test progress. Mice tested with peripheral and distal targets outperformed mice tested with a central target. A robust exploration pattern was identified in the probe test. COMPARISON WITH EXISTING METHOD: The MBM mimics natural environment to a higher degree of accuracy than the BM, without eliciting bias towards non-spatial searching strategies. CONCLUSIONS: Spatial learning in the MBM is a target-location sensitive process, providing flexibility in task difficulty. Along with overcoming biases towards non-spatial strategies, the MBM represents an improvement over the well-validated BM.

L-Lactate Promotes Adult Hippocampal Neurogenesis.

Neurogenesis, the formation of new neurons in the adult brain, is important for memory formation and extinction. One of the most studied external interventions that affect the rate of adult neurogenesis is physical exercise. Physical exercise promotes adult neurogenesis via several factors including brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Here, we identified L-lactate, a physical exercise-induced metabolite, as a factor that promotes adult hippocampal neurogenesis. While prolonged exposure to L-lactate promoted neurogenesis, no beneficial effect was exerted on cognitive learning and memory. Systemic pharmacological blocking of monocarboxylate transporter 2 (MCT2), which transports L-lactate to the brain, prevented lactate-induced neurogenesis, while 3,5-dihydroxybenzoic acid (3,5-DHBA), an agonist for the lactate-receptor hydroxycarboxylic acid receptor 1 (HCAR1), did not affect adult neurogenesis. These data suggest that L-lactate partially mediates the effect of physical exercise on adult neurogenesis, but not cognition, in a MCT2-dependent manner.

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.

Dopamine receptors in the rat entopeduncular nucleus.

Dopamine is critical for the normal functioning of the basal ganglia, modulating both input and output nuclei of this system. The distribution and function of each of the five dopamine receptor subtypes have been studied extensively in the striatum. However, the role of extrastriatal dopamine receptors in basal ganglia information processing is less clear. Here, we studied the anatomical distribution of dopamine receptors in one of the output nuclei of the rodent basal ganglia, the entopeduncular nucleus (EP). The presence of all dopamine receptor subtypes was verified in the EP using immunostaining. We detected co-localization of dopamine receptors with VGAT, which suggests presynaptic expression on GABAergic terminals. D1R and D2R were strongly colocalized with VGAT, whereas DR3-5 showed only sparse co-localization. We further labeled striatal or pallidal neurons with GFP and showed that only D1 receptors were co-localized with striatal terminals, while only D2R and D3R were co-localized with pallidal terminals. Dopamine receptors were also strongly co-localized with MAP2, indicating postsynaptic expression. Overall, these findings suggest that the dopaminergic system modulates activity in the EP both directly via postsynaptic receptors, and indirectly via GABAergic synapses stemming from the direct and indirect pathways.

A protocol for quantitative analysis of murine and human amyloid-ß1-40 and 1-42.

BACKGROUND: Amyloid-ß (Aß), a hallmark of Alzheimer's disease (AD), has long been a focus of basic and translation research in AD. Quantification and dissociation of the Aß fractions in their soluble and insoluble forms, is a key factor in numerous AD studies. NEW METHOD: Here we provide a generalized sandwich-enzyme-linked-immuno-sorbent-assay (sELISA) protocol for quantification of human and murine Aß1-40 and Aß1-42 and dissociation of these peptides to their soluble-oligomeric and insoluble-fibrillar forms. RESULTS: We have validated the levels of soluble and insoluble Aß1-40 and Aß1-42 in the 5XFAD AD and the Ts65Dn Down-Syndrome (DS) mouse models in both the cortex, hippocampus and blood as follows: (1) blood levels of Aß1-40 and Aß1-42 are elevated in both mouse strains. (2) 5XFAD mice exhibit elevated soluble and insoluble Aß1-40 in cortical and hippocampal tissues, soluble Aß1-42 in the hippocampus, and insoluble Aß1-42in both cortical and hippocampal tissues (3) Ts65Dn mice exhibit elevated levels of Aß1-40 in the cortex. COMPARISON WITH EXISTING METHODS: Several methodologies have been proposed for the high throughput measure of Aß, including HPLC-mass-spectrometry, micro-immunoelectrodes, immunoprecipitation and ELISA. Although commercial sELISA kits are widely used, herein, we describe a more accessible and cost-effective in-house protocol enabling to measure either human or murine, soluble and insoluble Aß1-40 and Aß1-42 levels. CONCLUSIONS: We provide a streamlined and accessible protocol for the assessment of soluble and insoluble Aß1-40 and Aß1-42 levels from mouse or human origins, enabling a higher accessibility for researchers in the field to generate reliable Aß-related measurements.

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.

Sirt6 alters adult hippocampal neurogenesis.

Sirtuins are pleiotropic NAD+ dependent histone deacetylases involved in metabolism, DNA damage repair, inflammation and stress resistance. SIRT6, a member of the sirtuin family, regulates the process of normal aging and increases the lifespan of male mice over-expressing Sirt6 by 15%. Neurogenesis, the formation of new neurons within the hippocampus of adult mammals, involves several complex stages including stem cell proliferation, differentiation, migration and network integration. During aging, the number of newly generated neurons continuously declines, and this is correlated with a decline in neuronal plasticity and cognitive behavior. In this study we investigated the involvement of SIRT6 in adult hippocampal neurogenesis. Mice over-expressing Sirt6 exhibit increased numbers of young neurons and decreased numbers of mature neurons, without affecting glial differentiation. This implies of an involvement of SIRT6 in neuronal differentiation and maturation within the hippocampus. This work adds to the expanding body of knowledge on the regulatory mechanisms underlying adult hippocampal neurogenesis, and describes novel roles for SIRT6 as a regulator of cell fate during adult hippocampal neurogenesis.

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.

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.

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.

Neuron-specific expression of tomosyn1 in the mouse hippocampal dentate gyrus impairs spatial learning and memory.

Tomosyn, a syntaxin-binding protein, is known to inhibit vesicle priming and synaptic transmission via interference with the formation of SNARE complexes. Using a lentiviral vector, we specifically overexpressed tomosyn1 in hippocampal dentate gyrus neurons in adult mice. Mice were then subjected to spatial learning and memory tasks and electrophysiological measurements from hippocampal slices. Tomosyn1-overexpression significantly impaired hippocampus-dependent spatial memory while tested in the Morris water maze. Further, tomosyn1-overexpressing mice utilize swimming strategies of lesser cognitive ability in the Morris water maze compared with control mice. Electrophysiological measurements at mossy fiber-CA3 synapses revealed impaired paired-pulse facilitation in the mossy fiber of tomosyn1-overexpressing mice. This study provides evidence for novel roles for tomosyn1 in hippocampus-dependent spatial learning and memory, potentially via decreased synaptic transmission in mossy fiber-CA3 synapses. Moreover, it provides new insight regarding the role of the hippocampal dentate gyrus and mossy fiber-CA3 synapses in swimming strategy preference, and in learning and memory.

Evidence for a developmental role for TLR4 in learning and memory.

Toll-like receptors (TLRs) play essential roles in innate immunity and increasing evidence indicates that these receptors are expressed in neurons, astrocytes and microglia in the brain where they mediate responses to infection, stress and injury. Very little is known about the roles of TLRs in cognition. To test the hypothesis that TLR4 has a role in hippocampus-dependent spatial learning and memory, we used mice deficient for TLR4 and mice receiving chronic TLR4 antagonist infusion to the lateral ventricles in the brain. We found that developmental TLR4 deficiency enhances spatial reference memory acquisition and memory retention, impairs contextual fear-learning and enhances motor functions, traits that were correlated with CREB up-regulation in the hippocampus. TLR4 antagonist infusion into the cerebral ventricles of adult mice did not affect cognitive behavior, but instead affected anxiety responses. Our findings indicate a developmental role for TLR4 in shaping spatial reference memory, and fear learning and memory. Moreover, we show that central TLR4 inhibition using a TLR4 antagonist has no discernible physiological role in regulating spatial and contextual hippocampus-dependent cognitive behavior.

Localization of molecular correlates of memory consolidation to buccal ganglia mechanoafferent neurons after learning that food is inedible in Aplysia.

Training paradigms affecting Aplysia withdrawal reflexes cause changes in gene expression leading to long-term memory formation in primary mechanoafferents that initiate withdrawal. Similar mechanoafferents are also found in the buccal ganglia that control feeding behavior, raising the possibility that these mechanoafferents are a locus of memory formation after a training paradigm affecting feeding. Buccal ganglia mechanoafferent neurons expressed increases in mRNA expression for the transcription factor ApC/EBP, and for the growth factor sensorin-A, within the first 2 h after training with an inedible food. No increases in expression were detected in the rest of the buccal ganglia. Increased ApC/EBP expression was not elicited by food and feeding responses not causing long-term memory. Increased ApC/EBP expression was directly related to a measure of the efficacy of training in causing long-term memory, suggesting that ApC/EBP expression is necessary for the expression of aspects of long-term memory. In behaving animals, memory is expressed as a decrease in the likelihood to respond to food, and a decrease in the amplitude of protraction, the first phase of consummatory feeding behaviors. To determine how changes in the properties of mechanoafferents could cause learned changes in feeding behavior, synaptic contacts were mapped from the mechanoafferents to the B31/B32 neurons, which have a key role in initiating consummatory behaviors and also control protractions. Many mechanoafferents monosynaptically and polysynaptically connect with B31/B32. Monosynaptic connections were complex combinations of fast and slow excitation and/or inhibition. Changes in the response of B31/B32 to stimuli sensed by the mechanoafferent could underlie aspects of long-term memory expression.

Autaptic muscarinic self-excitation and nitrergic self-inhibition in neurons initiating Aplysia feeding are revealed when the neurons are cultured in isolation.

Properties of a neuron may arise via endogenous mechanisms, or via interactions with other neurons. Culturing a neuron in isolation is a useful tool to distinguish between endogenous and circuit-derived properties. We identified two remarkable functional features of pattern initiator neurons B31/B32 in Aplysia when these neurons were cultured in isolation. These features were also present in situ, but were less prominent, and would have been missed had they not been observed first in the isolated cultured neurons. The properties are likely to be present in neurons of higher animals, but have not yet been observed. One feature was autaptic muscarinic self-excitation that contributes to the neuron's plateau potential, by which it initiates behavior. The other feature was the release of nitric oxide (NO) in the absence of spiking, which causes self-inhibition at rest. The nitrergic modulation of B31/B32 is likely to contribute to the control of feeding by dietary changes in the concentration of L: -arginine, the precursor from which NO is synthesized.