ABSTRACT
The histone lysine demethylase KDM5B is implicated in recessive intellectual disability disorders, and heterozygous, protein-truncating variants in KDM5B are associated with reduced cognitive function in the population. The KDM5 family of lysine demethylases has developmental and homeostatic functions in the brain, some of which appear to be independent of lysine demethylase activity. To determine the functions of KDM5B in hippocampus-dependent learning and memory, we first studied male and female mice homozygous for a Kdm5b Δ ARID allele that lacks demethylase activity. Kdm5b Δ ARID/ Δ ARID mice exhibited hyperactivity and long-term memory deficits in hippocampus-dependent learning tasks. The expression of immediate early, activity-dependent genes was downregulated in these mice and hyperactivated upon a learning stimulus compared with wild-type (WT) mice. A number of other learning-associated genes were also significantly dysregulated in the Kdm5b Δ ARID/ Δ ARID hippocampus. Next, we knocked down Kdm5b specifically in the adult, WT mouse hippocampus with shRNA. Kdm5b knockdown resulted in spontaneous seizures, hyperactivity, and hippocampus-dependent long-term memory and long-term potentiation deficits. These findings identify KDM5B as a critical regulator of gene expression and synaptic plasticity in the adult hippocampus and suggest that at least some of the cognitive phenotypes associated with KDM5B gene variants are caused by direct effects on memory consolidation mechanisms.
Subject(s)
Hippocampus , Intellectual Disability , Jumonji Domain-Containing Histone Demethylases , Memory Consolidation , Memory, Long-Term , Animals , Hippocampus/metabolism , Mice , Male , Female , Intellectual Disability/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Memory Consolidation/physiology , Memory, Long-Term/physiology , Long-Term Potentiation/genetics , Long-Term Potentiation/physiology , Mice, Inbred C57BL , DNA-Binding ProteinsABSTRACT
Technological advancements have facilitated the implementation of realistic, terrestrial-based complex 33-beam galactic cosmic radiation simulations (GCR Sim) to now probe central nervous system functionality. This work expands considerably on prior, simplified GCR simulations, yielding new insights into responses of male and female mice exposed to 40-50 cGy acute or chronic radiations relevant to deep space travel. Results of the object in updated location task suggested that exposure to acute or chronic GCR Sim induced persistent impairments in hippocampus-dependent memory formation and reconsolidation in female mice that did not manifest robustly in irradiated male mice. Interestingly, irradiated male mice, but not females, were impaired in novel object recognition and chronically irradiated males exhibited increased aggressive behavior on the tube dominance test. Electrophysiology studies used to evaluate synaptic plasticity in the hippocampal CA1 region revealed significant reductions in long-term potentiation after each irradiation paradigm in both sexes. Interestingly, network-level disruptions did not translate to altered intrinsic electrophysiological properties of CA1 pyramidal cells, whereas acute exposures caused modest drops in excitatory synaptic signaling in males. Ultrastructural analyses of CA1 synapses found smaller postsynaptic densities in larger spines of chronically exposed mice compared to controls and acutely exposed mice. Myelination was also affected by GCR Sim with acutely exposed mice exhibiting an increase in the percent of myelinated axons; however, the myelin sheathes on small calibur (< 0.3 mm) and larger (> 0.5 mm) axons were thinner when compared to controls. Present findings might have been predicted based on previous studies using single and mixed beam exposures and provide further evidence that space-relevant radiation exposures disrupt critical cognitive processes and underlying neuronal network-level plasticity, albeit not to the extent that might have been previously predicted.
Subject(s)
Hippocampus , Radiation Exposure , Female , Mice , Male , Animals , Synapses , Long-Term Potentiation , Neuronal PlasticityABSTRACT
INTRODUCTION: Synaptic loss is a hallmark of Alzheimer's disease (AD) that correlates with cognitive decline in AD patients. Complement-mediated synaptic pruning has been associated with this excessive loss of synapses in AD. Here, we investigated the effect of C5aR1 inhibition on microglial and astroglial synaptic pruning in two mouse models of AD. METHODS: A combination of super-resolution and confocal and tridimensional image reconstruction was used to assess the effect of genetic ablation or pharmacological inhibition of C5aR1 on the Arctic48 and Tg2576 models of AD. RESULTS: Genetic ablation or pharmacological inhibition of C5aR1 partially rescues excessive pre-synaptic pruning and synaptic loss in an age and region-dependent fashion in two mouse models of AD, which correlates with improved long-term potentiation (LTP). DISCUSSION: Reduction of excessive synaptic pruning is an additional beneficial outcome of the suppression of C5a-C5aR1 signaling, further supporting its potential as an effective targeted therapy to treat AD. HIGHLIGHTS: C5aR1 ablation restores long-term potentiation in the Arctic model of AD. C5aR1 ablation rescues region specific excessive pre-synaptic loss. C5aR1 antagonist, PMX205, rescues VGlut1 loss in the Tg2576 model of AD. C1q tagging is not sufficient to induce VGlut1 microglial ingestion. Astrocytes contribute to excessive pre-synaptic loss at late stages of the disease.
Subject(s)
Alzheimer Disease , Mice , Animals , Humans , Alzheimer Disease/genetics , Synapses , Long-Term Potentiation , Disease Models, AnimalABSTRACT
INTRODUCTION: The BIN1 coding variant rs138047593 (K358R) is linked to Late-Onset Alzheimer's Disease (LOAD) via targeted exome sequencing. METHODS: To elucidate the functional consequences of this rare coding variant on brain amyloidosis and neuroinflammation, we generated BIN1K358R knock-in mice using CRISPR/Cas9 technology. These mice were subsequently bred with 5xFAD transgenic mice, which serve as a model for Alzheimer's pathology. RESULTS: The presence of the BIN1K358R variant leads to increased cerebral amyloid deposition, with a dampened response of astrocytes and oligodendrocytes, but not microglia, at both the cellular and transcriptional levels. This correlates with decreased neurofilament light chain in both plasma and brain tissue. Synaptic densities are significantly increased in both wild-type and 5xFAD backgrounds homozygous for the BIN1K358R variant. DISCUSSION: The BIN1 K358R variant modulates amyloid pathology in 5xFAD mice, attenuates the astrocytic and oligodendrocytic responses to amyloid plaques, decreases damage markers, and elevates synaptic densities. HIGHLIGHTS: BIN1 rs138047593 (K358R) coding variant is associated with increased risk of LOAD. BIN1 K358R variant increases amyloid plaque load in 12-month-old 5xFAD mice. BIN1 K358R variant dampens astrocytic and oligodendrocytic response to plaques. BIN1 K358R variant decreases neuronal damage in 5xFAD mice. BIN1 K358R upregulates synaptic densities and modulates synaptic transmission.
Subject(s)
Alzheimer Disease , Animals , Mice , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid beta-Peptides , Disease Models, Animal , Mice, Transgenic , Neuroglia/pathology , Plaque, Amyloid/pathology , HumansABSTRACT
A recognized risk of long-duration space travel arises from the elevated exposure astronauts face from galactic cosmic radiation (GCR), which is composed of a diverse array of energetic particles. There is now abundant evidence that exposures to many different charged particle GCR components within acute time frames are sufficient to induce central nervous system deficits that span from the molecular to the whole animal behavioral scale. Enhanced spacecraft shielding can lessen exposures to charged particle GCR components, but may conversely elevate neutron radiation levels. We previously observed that space-relevant neutron radiation doses, chronically delivered at dose-rates expected during planned human exploratory missions, can disrupt hippocampal neuronal excitability, perturb network long-term potentiation and negatively impact cognitive behavior. We have now determined that acute exposures to similar low doses (18 cGy) of neutron radiation can also lead to suppressed hippocampal synaptic signaling, as well as decreased learning and memory performance in male mice. Our results demonstrate that similar nervous system hazards arise from neutron irradiation regardless of the exposure time course. While not always in an identical manner, neutron irradiation disrupts many of the same central nervous system elements as acute charged particle GCR exposures. The risks arising from neutron irradiation are therefore important to consider when determining the overall hazards astronauts will face from the space radiation environment.
Subject(s)
Cosmic Radiation/adverse effects , Hippocampus/radiation effects , Neutrons/adverse effects , Animals , Behavior, Animal/radiation effects , Male , Memory/radiation effects , Mice , Neuronal Plasticity/radiation effectsABSTRACT
Epigenetic mechanisms result in persistent changes at the cellular level that can lead to long-lasting behavioral adaptations. Nucleosome remodeling is a major epigenetic mechanism that has not been well explored with regards to drug-seeking behaviors. Nucleosome remodeling is performed by multi-subunit complexes that interact with DNA or chromatin structure and possess an ATP-dependent enzyme to disrupt nucleosome-DNA contacts and ultimately regulate gene expression. Calcium responsive transactivator (CREST) is a transcriptional activator that interacts with enzymes involved in both histone acetylation and nucleosome remodeling. Here, we examined the effects of knocking down CREST in the nucleus accumbens (NAc) core on drug-seeking behavior and synaptic plasticity in male mice as well as drug-seeking in male rats. Knocking down CREST in the NAc core results in impaired cocaine-induced conditioned place preference (CPP) as well as theta-induced long-term potentiation in the NAc core. Further, similar to the CPP findings, using a self-administration procedure, we found that CREST knockdown in the NAc core of male rats had no effect on instrumental responding for cocaine itself on a first-order schedule, but did significantly attenuate responding on a second-order chain schedule, in which responding has a weaker association with cocaine. Together, these results suggest that CREST in the NAc core is required for cocaine-induced CPP, synaptic plasticity, as well as cocaine-seeking behavior.SIGNIFICANCE STATEMENT This study demonstrates a key role for the role of Calcium responsive transactivator (CREST), a transcriptional activator, in the nucleus accumbens (NAc) core with regard to cocaine-induced conditioned place preference (CPP), self-administration (SA), and synaptic plasticity. CREST is a unique transcriptional regulator that can recruit enzymes from two different major epigenetic mechanisms: histone acetylation and nucleosome remodeling. In this study we also found that the level of potentiation in the NAc core correlated with whether or not animals formed a CPP. Together the results indicate that CREST is a key downstream regulator of cocaine action in the NAc.
Subject(s)
Cocaine/administration & dosage , Conditioning, Operant/physiology , Drug-Seeking Behavior/physiology , Neuronal Plasticity/physiology , Nucleus Accumbens/metabolism , Trans-Activators/biosynthesis , Animals , Conditioning, Operant/drug effects , Male , Mice , Mice, Inbred C57BL , Neuronal Plasticity/drug effects , Nucleus Accumbens/drug effects , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Long-Evans , Trans-Activators/deficiency , Trans-Activators/geneticsABSTRACT
Multiple epigenetic mechanisms, including histone acetylation and nucleosome remodeling, are known to be involved in long-term memory formation. Enhancing histone acetylation by deleting histone deacetylases, like HDAC3, typically enhances long-term memory formation. In contrast, disrupting nucleosome remodeling by blocking the neuron-specific chromatin remodeling subunit BAF53b impairs long-term memory. Here, we show that deleting HDAC3 can ameliorate the impairments in both long-term memory and synaptic plasticity caused by BAF53b mutation. This suggests a dynamic interplay exists between histone acetylation/deacetylation and nucleosome remodeling mechanisms in the regulation of memory formation.
Subject(s)
Histone Deacetylases/deficiency , Long-Term Potentiation/physiology , Memory Disorders/metabolism , Memory, Long-Term/physiology , Animals , Epigenesis, Genetic , Female , Hippocampus/metabolism , Histone Deacetylases/genetics , Long-Term Potentiation/genetics , Male , Memory Disorders/genetics , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
Dendritic extension and synaptogenesis proceed at high rates in rat hippocampus during early postnatal life but markedly slow during the third week of development. The reasons for the latter, fundamental event are poorly understood. Here, we report that levels of phosphorylated (inactive) cofilin, an actin depolymerizing factor, decrease by 90% from postnatal days (pnds) 10 to 21. During the same period, levels of total and phosphorylated Arp2, which nucleates actin branches, increase. A search for elements that could explain the switch from inactive to active cofilin identified reductions in ß1 integrin, TrkB, and LIM domain kinase 2b, upstream proteins that promote cofilin phosphorylation. Moreover, levels of slingshot 3, which dephosphorylates cofilin, increase during the period in which growth slows. Consistent with the cofilin results, in situ phalloidin labeling of F-actin demonstrated that spines and dendrites contained high levels of dynamic actin filaments during Week 2, but these fell dramatically by pnd 21. The results suggest that the change from inactive to constitutively active cofilin leads to a loss of dynamic actin filaments needed for process extension and thus the termination of spine formation and synaptogenesis. The relevance of these events to the emergence of memory-related synaptic plasticity is described.
Subject(s)
Actin Depolymerizing Factors/metabolism , Hippocampus/growth & development , Hippocampus/metabolism , Neurogenesis/physiology , Neuronal Plasticity/physiology , Animals , Blotting, Western , Immunohistochemistry , Immunoprecipitation , Male , Patch-Clamp Techniques , Rats , Rats, Sprague-DawleyABSTRACT
Recent human exome-sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several intellectual disabilities and cognitive disorders, including autism. However, it remains unclear how mutations in BAF complexes result in impaired cognitive function. Post-mitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Subdomain 2 of BAF53b is essential for the differentiation of neuronal precursor cells into neurons. We generated transgenic mice lacking subdomain 2 of Baf53b (BAF53bΔSB2). Long-term synaptic potentiation (LTP) and long-term memory, both of which are associated with phosphorylation of the actin severing protein cofilin, were assessed in these animals. A phosphorylation mimic of cofilin was stereotaxically delivered into the hippocampus of BAF53bΔSB2 mice in an effort to rescue LTP and memory. BAF53bΔSB2 mutant mice show impairments in phosphorylation of synaptic cofilin, LTP, and memory. Both the synaptic plasticity and memory deficits are rescued by overexpression of a phosphorylation mimetic of cofilin. Baseline physiology and behavior were not affected by the mutation or the experimental treatment. This study suggests a potential link between nBAF function, actin cytoskeletal remodeling at the dendritic spine, and memory formation. This work shows that a targeted manipulation of synaptic function can rescue adult plasticity and memory deficits caused by manipulations of nBAF, and thereby provides potential novel avenues for therapeutic development for multiple intellectual disability disorders.
Subject(s)
Chromatin Assembly and Disassembly/genetics , Chromosomal Proteins, Non-Histone/metabolism , Memory/physiology , Mutation/genetics , Neuronal Plasticity/genetics , Phosphopyruvate Hydratase/metabolism , Actin Depolymerizing Factors/genetics , Actin Depolymerizing Factors/metabolism , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Nucleolus/metabolism , Chromosomal Proteins, Non-Histone/genetics , Hippocampus/cytology , Hippocampus/metabolism , In Vitro Techniques , Long-Term Potentiation/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Net/physiology , Neurons/ultrastructure , Phosphopyruvate Hydratase/genetics , Phosphorylation/genetics , Sequence Deletion/genetics , Transduction, GeneticABSTRACT
Designer receptors exclusively activated by designer drug (DREADDs) are a novel tool with the potential to bidirectionally drive cellular, circuit, and ultimately, behavioral changes. We used DREADDs to evaluate memory formation in a hippocampus-dependent task in mice and effects on synaptic physiology in the dorsal hippocampus. We expressed neuron-specific (hSyn promoter) DREADDs that were either excitatory (HM3D) or inhibitory (HM4D) in the dorsal hippocampus. As predicted, hSyn-HM3D was able to transform a subthreshold learning event into long-term memory (LTM), and hSyn-HM4D completely impaired LTM formation. Surprisingly, the opposite was observed during experiments examining the effects on hippocampal long-term potentiation (LTP). hSyn-HM3D impaired LTP and hSyn-HM4D facilitated LTP. Follow-up experiments indicated that the hSyn-HM3D-mediated depression of fEPSP appears to be driven by presynaptic activation of inhibitory currents, whereas the hSyn-HM4D-mediated increase of fEPSP is induced by a reduction in GABAA receptor function. To determine whether these observations were promoter specific, we next examined the effects of using the CaMKIIα promoter that limits expression to forebrain excitatory neurons. CaMKIIα-HM3D in the dorsal hippocampus led to the transformation of a subthreshold learning event into LTM, whereas CaMKIIα-HM4D blocked LTM formation. Consistent with these findings, baseline synaptic transmission and LTP was increased in CaMKIIα-HM3D hippocampal slices, whereas slices from CaMKIIα-HM4D mice produced expected decreases in baseline synaptic transmission and LTP. Together, these experiments further demonstrate DREADDs as being a robust and reliable means of modulating neuronal function to manipulate long-term changes in behavior, while providing evidence for specific dissociations between LTM and LTP. SIGNIFICANCE STATEMENT: This study evaluates the efficacy of designer receptors exclusively activated by designer drug (DREADDs) as a means of bidirectionally modulating the hippocampus in not only a hippocampus-dependent task but also in hippocampal synaptic plasticity. This is the first study to evaluate the effects of DREADD-mediated inhibition and excitation in hippocampal long-term potentiation. More specifically, this study evaluates the effect of promoter-specific expression of DREADD viruses in a heterogenic cell population, which revealed surprising effects of different promoters. With chemogenetics becoming a more ubiquitous tool throughout studies investigating circuit-specific function, these data are of broad interest to the neuroscientific community because we have shown that promoter-specific effects can drastically alter synaptic function within a specific region, without parallel changes at the level of behavior.
Subject(s)
Designer Drugs/administration & dosage , Hippocampus/physiology , Long-Term Potentiation/physiology , Memory/physiology , Nerve Tissue Proteins/genetics , Neuronal Plasticity/physiology , Animals , Hippocampus/drug effects , Long-Term Potentiation/drug effects , Male , Memory/drug effects , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Neuronal Plasticity/drug effects , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , Radiation Dose Hypofractionation , Synaptic Transmission/drug effects , Synaptic Transmission/physiologyABSTRACT
KEY POINTS: Extended trains of theta rhythm afferent activity lead to a biphasic response facilitation in field CA1 but not in the lateral perforant path input to the dentate gyrus. Processes that reverse long-term potentiation in field CA1 are not operative in the lateral perforant path: multiple lines of evidence indicate that this reflects differences in adenosine signalling. Adenosine A1 receptors modulate baseline synaptic transmission in the lateral olfactory tract but not the associational afferents of the piriform cortex. Levels of ecto-5'-nucleotidase (CD73), an enzyme that converts extracellular ATP into adenosine, are markedly different between regions and correlate with adenosine signalling and the efficacy of theta pulse stimulation in reversing long-term potentiation. Variations in transmitter mobilization, CD73 levels, and afferent divergence result in multivariate differences in signal processing through nodes in the cortico-hippocampal network. ABSTRACT: The present study evaluated learning-related synaptic operations across the serial stages of the olfactory cortex-hippocampus network. Theta frequency stimulation produced very different time-varying responses in the Schaffer-commissural projections than in the lateral perforant path (LPP), an effect associated with distinctions in transmitter mobilization. Long-term potentiation (LTP) had a higher threshold in LPP field potential studies but not in voltage clamped neurons; coupled with input/output relationships, these results suggest that LTP threshold differences reflect the degree of input divergence. Theta pulse stimulation erased LTP in CA1 but not in the dentate gyrus (DG), although adenosine eliminated potentiation in both areas, suggesting that theta increases extracellular adenosine to a greater degree in CA1. Moreover, adenosine A1 receptor antagonism had larger effects on theta responses in CA1 than in the DG, and concentrations of ecto-5'-nucleotidase (CD73) were much higher in CA1. Input/output curves for two connections in the piriform cortex were similar to those for the LPP, whereas adenosine modulation again correlated with levels of CD73. In sum, multiple relays in a network extending from the piriform cortex through the hippocampus can be differentiated along three dimensions (input divergence, transmitter mobilization, adenosine modulation) that potently influence throughput and plasticity. A model that incorporates the regional differences, supplemented with data for three additional links, suggests that network output goes through three transitions during the processing of theta input. It is proposed that individuated relays allow the circuit to deal with different types of behavioural problems.
Subject(s)
Adenosine/metabolism , CA1 Region, Hippocampal/physiology , Long-Term Potentiation , Piriform Cortex/physiology , Synaptic Potentials , 5'-Nucleotidase/metabolism , Animals , CA1 Region, Hippocampal/metabolism , Dentate Gyrus/metabolism , Dentate Gyrus/physiology , Male , Piriform Cortex/metabolism , Rats , Rats, Sprague-Dawley , Theta RhythmABSTRACT
The superiority of spaced vs. massed training is a fundamental feature of learning. Here, we describe unanticipated timing rules for the production of long-term potentiation (LTP) in adult rat hippocampal slices that can account for one temporal segment of the spaced trials phenomenon. Successive bouts of naturalistic theta burst stimulation of field CA1 afferents markedly enhanced previously saturated LTP if spaced apart by 1 h or longer, but were without effect when shorter intervals were used. Analyses of F-actin-enriched spines to identify potentiated synapses indicated that the added LTP obtained with delayed theta trains involved recruitment of synapses that were "missed" by the first stimulation bout. Single spine glutamate-uncaging experiments confirmed that less than half of the spines in adult hippocampus are primed to undergo plasticity under baseline conditions, suggesting that intrinsic variability among individual synapses imposes a repetitive presentation requirement for maximizing the percentage of potentiated connections. We propose that a combination of local diffusion from initially modified spines coupled with much later membrane insertion events dictate that the repetitions be widely spaced. Thus, the synaptic mechanisms described here provide a neurobiological explanation for one component of a poorly understood, ubiquitous aspect of learning.
Subject(s)
Learning/physiology , Synapses/physiology , Actins/metabolism , Animals , Dendritic Spines/physiology , In Vitro Techniques , Long-Term Potentiation/physiology , Male , Mice , Mice, Inbred C57BL , Polymerization , Rats , Rats, Sprague-Dawley , Synaptic Transmission/physiology , Theta Rhythm/physiology , Time FactorsABSTRACT
Cranial irradiation used to control brain malignancies invariably leads to progressive and debilitating declines in cognition. Clinical efforts implementing hippocampal avoidance and NMDAR antagonism, have sought to minimize dose to radiosensitive neurogenic regions while normalizing excitatory/inhibitory (E/I) tone. Results of these trials have yielded only marginal benefits to cognition, prompting current studies to evaluate the potential of systemic extracellular vesicle (EV) therapy to restore neurocognitive functionality in the irradiated brain. Here we tested the hypothesis that EVs derived from inhibitory but not excitatory neuronal cultures would prove beneficial to cognition and associated pathology. Rats subjected to a clinically relevant, fractionated cranial irradiation paradigm were given multiple injections of either GABAergic- or glutamatergic-derived EV and subjected to behavioral testing. Rats treated with GABAergic but not glutamatergic EVs showed significant improvements on hippocampal- and cortical-dependent behavioral tasks. While each treatment enhanced levels of the neurotrophic factors BDNF and GDNF, only GABAergic EVs preserved granule cell neuron dendritic spine density. Additional studies conducted with GABAergic EVs, confirmed significant benefits on amygdala-dependent behavior and modest changes in synaptic plasticity as measured by long-term potentiation. These data point to a potentially more efficacious approach for resolving radiation-induced neurological deficits, possibly through a mechanism able to restore homeostatic E/I balance.
Subject(s)
Cranial Irradiation , Extracellular Vesicles , GABAergic Neurons , Animals , Extracellular Vesicles/metabolism , Rats , Cranial Irradiation/adverse effects , GABAergic Neurons/metabolism , GABAergic Neurons/radiation effects , Male , Hippocampus/radiation effects , Hippocampus/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Neurons/radiation effects , Neurons/metabolism , Glutamic Acid/metabolism , Neuronal Plasticity/radiation effects , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Behavior, Animal/radiation effectsABSTRACT
Gene-editing technologies promise to create a new class of therapeutics that can achieve permanent correction with a single intervention. Besides eliminating mutant alleles in familial disease, gene-editing can also be used to favorably manipulate upstream pathophysiologic events and alter disease-course in wider patient populations, but few such feasible therapeutic avenues have been reported. Here we use CRISPR-Cas9 to edit the last exon of amyloid precursor protein (App), relevant for Alzheimer's disease (AD). Our strategy effectively eliminates an endocytic (YENPTY) motif at APP C-terminus, while preserving the N-terminus and compensatory APP-homologues. This manipulation favorably alters events along the amyloid-pathway - inhibiting toxic APP-ß-cleavage fragments (including Aß) and upregulating neuroprotective APP-α-cleavage products. AAV-driven editing ameliorates neuropathologic, electrophysiologic, and behavioral deficits in an AD knockin mouse model. Effects persist for many months, and no abnormalities are seen in WT mice even after germline App-editing; underlining overall efficacy and safety. Pathologic alterations in the glial-transcriptome of App-KI mice, as seen by single nuclei RNA-sequencing (sNuc-Seq), are also normalized by App C-terminus editing. Our strategy takes advantage of innate transcriptional rules that render terminal exons insensitive to nonsense-decay, and the upstream manipulation is expected to be effective for all forms of AD. These studies offer a path for a one-time disease-modifying treatment for AD.
ABSTRACT
Exercise has beneficial effects on cognition throughout the lifespan. Here, we demonstrate that specific exercise patterns transform insufficient, subthreshold training into long-term memory in mice. Our findings reveal a potential molecular memory window such that subthreshold training within this window enables long-term memory formation. We performed RNA-seq on dorsal hippocampus and identify genes whose expression correlate with conditions in which exercise enables long-term memory formation. Among these genes we found Acvr1c, a member of the TGF ß family. We find that exercise, in any amount, alleviates epigenetic repression at the Acvr1c promoter during consolidation. Additionally, we find that ACVR1C can bidirectionally regulate synaptic plasticity and long-term memory in mice. Furthermore, Acvr1c expression is impaired in the aging human and mouse brain, as well as in the 5xFAD mouse model, and over-expression of Acvr1c enables learning and facilitates plasticity in mice. These data suggest that promoting ACVR1C may protect against cognitive impairment.
Subject(s)
Activin Receptors, Type I , Epigenesis, Genetic , Hippocampus , Memory, Long-Term , Physical Conditioning, Animal , Animals , Female , Humans , Male , Mice , Activin Receptors, Type I/genetics , Activin Receptors, Type I/metabolism , Aging/genetics , Aging/physiology , Hippocampus/metabolism , Memory, Long-Term/physiology , Mice, Inbred C57BL , Neuronal Plasticity/genetics , Physical Conditioning, Animal/physiology , Promoter Regions, GeneticABSTRACT
Evolving evidence suggests that brain inflammation and the buildup of proinflammatory cytokine increases the risk for cognitive decline and cognitive dysfunction. Interleukin-1ß (IL-1ß), acting via poorly understood mechanisms, appears to be a key cytokine in causing these deleterious effects along with a presumably related loss of long-term potentiation (LTP)-type synaptic plasticity. We hypothesized that IL-1ß disrupts brain-derived neurotrophic factor (BDNF) signaling cascades and thereby impairs the formation of filamentous actin (F-actin) in dendritic spines, an event that is essential for the stabilization of LTP. Actin polymerization in spines requires phosphorylation of the filament severing protein cofilin and is modulated by expression of the immediate early gene product Arc. Using rat organotypic hippocampal cultures, we found that IL-1ß suppressed BDNF-dependent regulation of Arc and phosphorylation of cofilin and cAMP response element-binding protein (CREB), a transcription factor regulating Arc expression. IL-1ß appears to act on BDNF signal transduction by impairing the phosphorylation of insulin receptor substrate 1, a protein that couples activation of the BDNF receptor TrkB to downstream signaling pathways regulating CREB, Arc, and cofilin. IL-1ß upregulated p38 mitogen-activated protein kinase (MAPK), and inhibiting p38 MAPK prevented IL-1ß from disrupting BDNF signaling. IL-1ß also prevented the formation of F-actin in spines and impaired the consolidation, but not the induction, of BDNF-dependent LTP in acute hippocampal slices. The suppressive effect of IL-1ß on F-actin and LTP was prevented by inhibiting p38 MAPK. These findings define a new mechanism for the action of IL-1ß on LTP and point to a potential therapeutic target to restore synaptic plasticity.
Subject(s)
Brain-Derived Neurotrophic Factor/antagonists & inhibitors , Interleukin-1beta/physiology , Neuronal Plasticity/physiology , p38 Mitogen-Activated Protein Kinases/physiology , Actins/metabolism , Animals , Brain-Derived Neurotrophic Factor/pharmacology , Brain-Derived Neurotrophic Factor/physiology , Cells, Cultured , Cerebral Cortex/metabolism , Cerebral Cortex/physiology , Cofilin 1/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cytoskeletal Proteins/biosynthesis , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Gene Expression Regulation/physiology , Hippocampus/physiology , Interleukin-1beta/pharmacology , Long-Term Potentiation/physiology , Male , Mitogen-Activated Protein Kinases/metabolism , Nerve Tissue Proteins/biosynthesis , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/physiology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Memory consolidation theory posits that newly acquired information passes through a series of stabilization steps before being firmly encoded. We report here that in rat and mouse, hippocampus cell adhesion receptors belonging to the ß1-integrin family exhibit dynamic properties in adult synapses and that these contribute importantly to a previously unidentified stage of consolidation. Quantitative dual immunofluorescence microscopy showed that induction of long-term potentiation (LTP) by theta burst stimulation (TBS) activates ß1 integrins, and integrin-signaling kinases, at spine synapses in adult hippocampal slices. Neutralizing antisera selective for ß1 integrins blocked these effects. TBS-induced integrin activation was brief (<7 min) and followed by an â¼45 min period during which the adhesion receptors did not respond to a second application of TBS. Brefeldin A, which blocks integrin trafficking to the plasma membrane, prevented the delayed recovery of integrin responses to TBS. ß1 integrin-neutralizing antisera erased LTP when applied during, but not after, the return of integrin responsivity. Similarly, infusions of anti-ß1 into rostral mouse hippocampus blocked formation of long-term, object location memory when started 20 min after learning but not 40 min later. The finding that ß1 integrin neutralization was effective in the same time window for slice and behavioral experiments strongly suggests that integrin recovery triggers a temporally discrete, previously undetected second stage of consolidation for both LTP and memory.
Subject(s)
Hippocampus/physiology , Integrin beta1/metabolism , Long-Term Potentiation/physiology , Memory/physiology , Neural Cell Adhesion Molecules/metabolism , Neuronal Plasticity/physiology , Animals , Male , Mice , Mice, Inbred C57BL , Rats , Rats, Sprague-DawleyABSTRACT
INTRODUCTION: Synaptic loss is a hallmark of Alzheimer's disease (AD) that correlates with cognitive decline in AD patients. Complement-mediated synaptic pruning has been associated with this excessive loss of synapses in AD. Here, we investigated the effect of C5aR1 inhibition on microglial and astroglial synaptic pruning in two mouse models of AD. METHODS: A combination of super-resolution and confocal and tridimensional image reconstruction was used to assess the effect of genetic ablation or pharmacological inhibition of C5aR1 on the Arctic48 and Tg2576 models of AD. RESULTS: Genetic ablation or pharmacological inhibition of C5aR1 rescues the excessive pre-synaptic pruning and synaptic loss in an age and region dependent fashion in two mouse models of AD, which correlates with improved long-term potentiation (LTP). DISCUSSION: Reduction of excessive synaptic pruning is an additional beneficial outcome of the suppression of C5a-C5aR1 signaling, further supporting its potential as an effective targeted therapy to treat AD.
ABSTRACT
Long-term potentiation (LTP) was used to gauge the impact of conventional and FLASH dose rates on synaptic transmission. Data collected from the hippocampus and medial prefrontal cortex confirmed significant inhibition of LTP after 10 fractions of 3 Gy (30 Gy total) conventional radiotherapy. Remarkably, 10x3Gy FLASH radiotherapy and unirradiated controls were identical and exhibited normal LTP.
Subject(s)
Long-Term Potentiation , Neuronal Plasticity , Mice , Animals , Neuronal Plasticity/physiology , Long-Term Potentiation/physiology , Hippocampus/physiology , Synaptic Transmission/physiologyABSTRACT
Implementation of ultra-high dose-rate FLASH radiotherapy (FLASH-RT) is rapidly gaining traction as a unique cancer treatment modality able to dramatically minimize normal tissue toxicity while maintaining antitumor efficacy compared with standard-of-care radiotherapy at conventional dose rate (CONV-RT). The resultant improvements in the therapeutic index have sparked intense investigations in pursuit of the underlying mechanisms. As a preamble to clinical translation, we exposed non-tumor-bearing male and female mice to hypofractionated (3 × 10 Gy) whole brain FLASH- and CONV-RT to evaluate differential neurologic responses using a comprehensive panel of functional and molecular outcomes over a 6-month follow-up. In each instance, extensive and rigorous behavioral testing showed FLASH-RT to preserve cognitive indices of learning and memory that corresponded to a similar protection of synaptic plasticity as measured by long-term potentiation (LTP). These beneficial functional outcomes were not found after CONV-RT and were linked to a preservation of synaptic integrity at the molecular (synaptophysin) level and to reductions in neuroinflammation (CD68+ microglia) throughout specific brain regions known to be engaged by our selected cognitive tasks (hippocampus, medial prefrontal cortex). Ultrastructural changes in presynaptic/postsynaptic bouton (Bassoon/Homer-1 puncta) within these same regions of the brain were not found to differ in response to dose rate. With this clinically relevant dosing regimen, we provide a mechanistic blueprint from synapse to cognition detailing how FLASH-RT reduces normal tissue complications in the irradiated brain. Significance: Functional preservation of cognition and LTP after hypofractionated FLASH-RT are linked to a protection of synaptic integrity and a reduction in neuroinflammation over protracted after irradiation times.