Microglia-synapse engulfment via PtdSer-TREM2 ameliorates neuronal hyperactivity in Alzheimer's disease models.

Neuronal hyperactivity is a key feature of early stages of Alzheimer's disease (AD). Genetic studies in AD support that microglia act as potential cellular drivers of disease risk, but the molecular determinants of microglia-synapse engulfment associated with neuronal hyperactivity in AD are unclear. Here, using super-resolution microscopy, 3D-live imaging of co-cultures, and in vivo imaging of lipids in genetic models, we found that spines become hyperactive upon Aß oligomer stimulation and externalize phosphatidylserine (ePtdSer), a canonical "eat-me" signal. These apoptotic-like spines are targeted by microglia for engulfment via TREM2 leading to amelioration of Aß oligomer-induced synaptic hyperactivity. We also show the in vivo relevance of ePtdSer-TREM2 signaling in microglia-synapse engulfment in the hAPP NL-F knock-in mouse model of AD. Higher levels of apoptotic-like synapses in mice as well as humans that carry TREM2 loss-of-function variants were also observed. Our work supports that microglia remove hyperactive ePtdSer+ synapses in Aß-relevant context and suggest a potential beneficial role for microglia in the earliest stages of AD.

Pinpointing the locus of GABAergic vulnerability in Alzheimer's disease.

The early stages of Alzheimer's disease (AD) have been linked to microcircuit dysfunction and pathophysiological neuronal firing in several brain regions. Inhibitory GABAergic microcircuitry is a critical feature of stable neural-circuit function in the healthy brain, and its dysregulation has therefore been proposed as contributing to AD-related pathophysiology. However, exactly how the critical balance between excitatory and inhibitory microcircuitry is modified by AD pathogenesis remains unclear. Here, we set the current evidence implicating dysfunctional GABAergic microcircuitry as a driver of early AD pathophysiology in a simple conceptual framework. Our framework is based on a generalised reductionist model of firing-rate control by local feedback inhibition. We use this framework to consider multiple loci that may be vulnerable to disruption by AD pathogenesis. We first start with evidence investigating how AD-related processes may impact the gross number of inhibitory neurons in the network. We then move to discuss how pathology may impact intrinsic cellular properties and firing thresholds of GABAergic neurons. Finally, we cover how AD-related pathogenesis may disrupt synaptic connectivity between excitatory and inhibitory neurons. We use the feedback inhibition framework to discuss and organise the available evidence from both preclinical rodent work and human studies in AD patients and conclude by identifying key questions and understudied areas for future investigation.

Quantitative super-resolution imaging of pathological aggregates reveals distinct toxicity profiles in different synucleinopathies.

Protein aggregation is a hallmark of major neurodegenerative disorders. Increasing data suggest that smaller aggregates cause higher toxic response than filamentous aggregates (fibrils). However, the size of small aggregates has challenged their detection within biologically relevant environments. Here, we report approaches to quantitatively super-resolve aggregates in live cells and ex vivo brain tissues. We show that Amytracker 630 (AT630), a commercial aggregate-activated fluorophore, has outstanding photophysical properties that enable super-resolution imaging of α-synuclein, tau, and amyloid-ß aggregates, achieving ∼4 nm precision. Applying AT630 to AppNL-G-F mouse brain tissues or aggregates extracted from a Parkinson's disease donor, we demonstrate excellent agreement with antibodies specific for amyloid-ß or α-synuclein, respectively, confirming the specificity of AT630. Subsequently, we use AT630 to reveal a linear relationship between α-synuclein aggregate size and cellular toxicity and discovered that aggregates smaller than 450 ± 60 nm (aggregate450nm) readily penetrated the plasma membrane. We determine aggregate450nm concentrations in six Parkinson's disease and dementia with Lewy bodies donor samples and show that aggregates in different synucleinopathies demonstrate distinct potency in toxicity. We further show that cell-penetrating aggregates are surrounded by proteasomes, which assemble into foci to gradually process aggregates. Our results suggest that the plasma membrane effectively filters out fibrils but is vulnerable to penetration by aggregates of 450 ± 60 nm. Together, our findings present an exciting strategy to determine specificity of aggregate toxicity within heterogeneous samples. Our approach to quantitatively measure these toxic aggregates in biological environments opens possibilities to molecular examinations of disease mechanisms under physiological conditions.

Metabolome-wide association study on ABCA7 indicates a role of ceramide metabolism in Alzheimer's disease.

Genome-wide association studies (GWASs) have identified genetic loci associated with the risk of Alzheimer's disease (AD), but the molecular mechanisms by which they confer risk are largely unknown. We conducted a metabolome-wide association study (MWAS) of AD-associated loci from GWASs using untargeted metabolic profiling (metabolomics) by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). We identified an association of lactosylceramides (LacCer) with AD-related single-nucleotide polymorphisms (SNPs) in ABCA7 (P = 5.0 × 10-5 to 1.3 × 10-44). We showed that plasma LacCer concentrations are associated with cognitive performance and genetically modified levels of LacCer are associated with AD risk. We then showed that concentrations of sphingomyelins, ceramides, and hexosylceramides were altered in brain tissue from Abca7 knockout mice, compared with wild type (WT) (P = 0.049-1.4 × 10-5), but not in a mouse model of amyloidosis. Furthermore, activation of microglia increases intracellular concentrations of hexosylceramides in part through induction in the expression of sphingosine kinase, an enzyme with a high control coefficient for sphingolipid and ceramide synthesis. Our work suggests that the risk for AD arising from functional variations in ABCA7 is mediated at least in part through ceramides. Modulation of their metabolism or downstream signaling may offer new therapeutic opportunities for AD.

Pharmacological ablation of astrocytes reduces Aß degradation and synaptic connectivity in an ex vivo model of Alzheimer's disease.

BACKGROUND: Astrocytes provide a vital support to neurons in normal and pathological conditions. In Alzheimer's disease (AD) brains, reactive astrocytes have been found surrounding amyloid plaques, forming an astrocytic scar. However, their role and potential mechanisms whereby they affect neuroinflammation, amyloid pathology, and synaptic density in AD remain unclear. METHODS: To explore the role of astrocytes on Aß pathology and neuroinflammatory markers, we pharmacologically ablated them in organotypic brain culture slices (OBCSs) from 5XFAD mouse model of AD and wild-type (WT) littermates with the selective astrocytic toxin L-alpha-aminoadipate (L-AAA). To examine the effects on synaptic circuitry, we measured dendritic spine number and size in OBCSs from Thy-1-GFP transgenic mice incubated with synthetic Aß42 or double transgenics Thy-1-GFP/5XFAD mice treated with LAAA or vehicle for 24 h. RESULTS: Treatment of OBCSs with L-AAA resulted in an increased expression of pro-inflammatory cytokine IL-6 in conditioned media of WTs and 5XFAD slices, associated with changes in microglia morphology but not in density. The profile of inflammatory markers following astrocytic loss was different in WT and transgenic cultures, showing reductions in inflammatory mediators produced in astrocytes only in WT sections. In addition, pharmacological ablation of astrocytes led to an increase in Aß levels in homogenates of OBCS from 5XFAD mice compared with vehicle controls, with reduced enzymatic degradation of Aß due to lower neprilysin and insulin-degrading enzyme (IDE) expression. Furthermore, OBSCs from wild-type mice treated with L-AAA and synthetic amyloid presented 56% higher levels of Aß in culture media compared to sections treated with Aß alone, concomitant with reduced expression of IDE in culture medium, suggesting that astrocytes contribute to Aß clearance and degradation. Quantification of hippocampal dendritic spines revealed a reduction in their density following L-AAA treatment in all groups analyzed. In addition, pharmacological ablation of astrocytes resulted in a decrease in spine size in 5XFAD OBCSs but not in OBCSs from WT treated with synthetic Aß compared to vehicle control. CONCLUSIONS: Astrocytes play a protective role in AD by aiding Aß clearance and supporting synaptic plasticity.

Nonlinear transfer of signal and noise correlations in cortical networks.

Signal and noise correlations, a prominent feature of cortical activity, reflect the structure and function of networks during sensory processing. However, in addition to reflecting network properties, correlations are also shaped by intrinsic neuronal mechanisms. Here we show that spike threshold transforms correlations by creating nonlinear interactions between signal and noise inputs; even when input noise correlation is constant, spiking noise correlation varies with both the strength and correlation of signal inputs. We characterize these effects systematically in vitro in mice and demonstrate their impact on sensory processing in vivo in gerbils. We also find that the effects of nonlinear correlation transfer on cortical responses are stronger in the synchronized state than in the desynchronized state, and show that they can be reproduced and understood in a model with a simple threshold nonlinearity. Since these effects arise from an intrinsic neuronal property, they are likely to be present across sensory systems and, thus, our results are a critical step toward a general understanding of how correlated spiking relates to the structure and function of cortical networks.

Delayed and Temporally Imprecise Neurotransmission in Reorganizing Cortical Microcircuits.

Synaptic neurotransmission is modified at cortical connections throughout life. Varying the amplitude of the postsynaptic response is one mechanism that generates flexible signaling in neural circuits. The timing of the synaptic response may also play a role. Here, we investigated whether weakening and loss of an entire connection between excitatory cortical neurons was foreshadowed in the timing of the postsynaptic response. We made electrophysiological recordings in rat primary somatosensory cortex that was undergoing experience-dependent loss of complete local excitatory connections. The synaptic latency of pyramid-pyramid connections, which typically comprise multiple synapses, was longer and more variable. Connection strength and latency were not correlated. Instead, prolonged latency was more closely related to progression of connection loss. The action potential waveform and axonal conduction velocity were unaffected, suggesting that the altered timing of neurotransmission was attributable to a synaptic mechanism. Modeling studies indicated that increasing the latency and jitter at a subset of synapses reduced the number of action potentials fired by a postsynaptic neuron. We propose that prolonged synaptic latency and diminished temporal precision of neurotransmission are hallmarks of impending loss of a cortical connection.

Rapid Bidirectional Reorganization of Cortical Microcircuits.

Mature neocortex adapts to altered sensory input by changing neural activity in cortical circuits. The underlying cellular mechanisms remain unclear. We used blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to show reorganization in somatosensory cortex elicited by altered whisker sensory input. We found that there was rapid expansion followed by retraction of whisker cortical maps. The cellular basis for the reorganization in primary somatosensory cortex was investigated with paired electrophysiological recordings in the periphery of the expanded whisker representation. During map expansion, the chance of finding a monosynaptic connection between pairs of pyramidal neurons increased 3-fold. Despite the rapid increase in local excitatory connectivity, the average strength and synaptic dynamics did not change, which suggests that new excitatory connections rapidly acquire the properties of established excitatory connections. During map retraction, entire excitatory connections between pyramidal neurons were lost. In contrast, connectivity between pyramidal neurons and fast spiking interneurons was unchanged. Hence, the changes in local excitatory connectivity did not occur in all circuits involving pyramidal neurons. Our data show that pyramidal neurons are recruited to and eliminated from local excitatory networks over days. These findings suggest that the local excitatory connectome is dynamic in mature neocortex.

Pansynaptic enlargement at adult cortical connections strengthened by experience.

Behavioral experience alters the strength of neuronal connections in adult neocortex. These changes in synaptic strength are thought to be central to experience-dependent plasticity, learning, and memory. However, it is not known how changes in synaptic transmission between neurons become persistent, thereby enabling the storage of previous experience. A long-standing hypothesis is that altered synaptic strength is maintained by structural modifications to synapses. However, the extent of synaptic modifications and the changes in neurotransmission that the modifications support remain unclear. To address these questions, we recorded from pairs of synaptically connected layer 2/3 pyramidal neurons in the barrel cortex and imaged their contacts with high-resolution confocal microscopy after altering sensory experience by whisker trimming. Excitatory connections strengthened by experience exhibited larger axonal varicosities, dendritic spines, and interposed contact zones. Electron microscopy showed that contact zone size was strongly correlated with postsynaptic density area. Therefore, our findings indicate that whole synapses are larger at strengthened connections. Synaptic transmission was both stronger and more reliable following experience-dependent synapse enlargement. Hence, sensory experience modified both presynaptic and postsynaptic function. Our findings suggest that the enlargement of synaptic contacts is an integral part of long-lasting strengthening of cortical connections and, hence, of information storage in the neocortex.

Age-related dysregulation of homeostatic control in neuronal microcircuits.

Neuronal homeostasis prevents hyperactivity and hypoactivity. Age-related hyperactivity suggests homeostasis may be dysregulated in later life. However, plasticity mechanisms preventing age-related hyperactivity and their efficacy in later life are unclear. We identify the adult cortical plasticity response to elevated activity driven by sensory overstimulation, then test how plasticity changes with age. We use in vivo two-photon imaging of calcium-mediated cellular/synaptic activity, electrophysiology and c-Fos-activity tagging to show control of neuronal activity is dysregulated in the visual cortex in late adulthood. Specifically, in young adult cortex, mGluR5-dependent population-wide excitatory synaptic weakening and inhibitory synaptogenesis reduce cortical activity following overstimulation. In later life, these mechanisms are downregulated, so that overstimulation results in synaptic strengthening and elevated activity. We also find overstimulation disrupts cognition in older but not younger animals. We propose that specific plasticity mechanisms fail in later life dysregulating neuronal microcircuit homeostasis and that the age-related response to overstimulation can impact cognitive performance.

Translocator protein is a marker of activated microglia in rodent models but not human neurodegenerative diseases.

Microglial activation plays central roles in neuroinflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18 kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells depends on the transcription factor AP1 and is unique to a subset of rodent species within the Muroidea superfamily. Finally, we identify LCP2 and TFEC as potential markers of microglial activation in humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO-PET signals in humans reflect the density of inflammatory cells rather than activation state.

Homeostatic regulation through strengthening of neuronal network-correlated synaptic inputs.

Homeostatic regulation is essential for stable neuronal function. Several synaptic mechanisms of homeostatic plasticity have been described, but the functional properties of synapses involved in homeostasis are unknown. We used longitudinal two-photon functional imaging of dendritic spine calcium signals in visual and retrosplenial cortices of awake adult mice to quantify the sensory deprivation-induced changes in the responses of functionally identified spines. We found that spines whose activity selectively correlated with intrinsic network activity underwent tumor necrosis factor alpha (TNF-α)-dependent homeostatic increases in their response amplitudes, but spines identified as responsive to sensory stimulation did not. We observed an increase in the global sensory-evoked responses following sensory deprivation, despite the fact that the identified sensory inputs did not strengthen. Instead, global sensory-evoked responses correlated with the strength of network-correlated inputs. Our results suggest that homeostatic regulation of global responses is mediated through changes to intrinsic network-correlated inputs rather than changes to identified sensory inputs thought to drive sensory processing.

Learning and memory: Scaling new areas.

A new study explores the neural-circuit and synaptic processes that support the transition from general to specific aversive memory formation. A critical role for homeostatic synaptic down-scaling in shaping the specificity of an associative memory is identified.

The aging mouse brain: cognition, connectivity and calcium.

Aging is a complex process that differentially impacts multiple cognitive, sensory, neuronal and molecular processes. Technological innovations now allow for parallel investigation of neuronal circuit function, structure and molecular composition in the brain of awake behaving adult mice. Thus, mice have become a critical tool to better understand how aging impacts the brain. However, a more granular systems-based approach, which considers the impact of age on key features relating to neural processing, is required. Here, we review evidence probing the impact of age on the mouse brain. We focus on a range of processes relating to neuronal function, including cognitive abilities, sensory systems, synaptic plasticity and calcium regulation. Across many systems, we find evidence for prominent age-related dysregulation even before 12 months of age, suggesting that emerging age-related alterations can manifest by late adulthood. However, we also find reports suggesting that some processes are remarkably resilient to aging. The evidence suggests that aging does not drive a parallel, linear dysregulation of all systems, but instead impacts some processes earlier, and more severely, than others. We propose that capturing the more fine-scale emerging features of age-related vulnerability and resilience may provide better opportunities for the rejuvenation of the aged brain.

Multi-scale network imaging in a mouse model of amyloidosis.

The adult neocortex is not hard-wired but instead retains the capacity to reorganise across multiple spatial scales long into adulthood. Plastic reorganisation occurs at the level of mesoscopic sensory maps, functional neuronal assemblies and synaptic ensembles and is thought to be a critical feature of neuronal network function. Here, we describe a series of approaches that use calcium imaging to measure network reorganisation across multiple spatial scales in vivo. At the mesoscopic level, we demonstrate that sensory activity can be measured in animals undergoing longitudinal behavioural assessment involving automated touchscreen tasks. At the cellular level, we show that network dynamics can be longitudinally measured at both stable and transient functional assemblies. At the level of single synapses, we show that functional subcellular calcium imaging approaches can be used to measure synaptic ensembles of dendritic spines in vivo. Finally, we demonstrate that all three levels of imaging can be spatially related to local pathology in a preclinical rodent model of amyloidosis. We propose that multi-scale in vivo calcium imaging can be used to measure parallel plasticity processes operating across multiple spatial scales in both the healthy brain and preclinical models of disease.

Audio-visual experience strengthens multisensory assemblies in adult mouse visual cortex.

We experience the world through multiple senses simultaneously. To better understand mechanisms of multisensory processing we ask whether inputs from two senses (auditory and visual) can interact and drive plasticity in neural-circuits of the primary visual cortex (V1). Using genetically-encoded voltage and calcium indicators, we find coincident audio-visual experience modifies both the supra and subthreshold response properties of neurons in L2/3 of mouse V1. Specifically, we find that after audio-visual pairing, a subset of multimodal neurons develops enhanced auditory responses to the paired auditory stimulus. This cross-modal plasticity persists over days and is reflected in the strengthening of small functional networks of L2/3 neurons. We find V1 processes coincident auditory and visual events by strengthening functional associations between feature specific assemblies of multimodal neurons during bouts of sensory driven co-activity, leaving a trace of multisensory experience in the cortical network.

Size-Dependent Axonal Bouton Dynamics following Visual Deprivation In Vivo.

Persistent synapses are thought to underpin the storage of sensory experience, yet little is known about their structural plasticity in vivo. We investigated how persistent presynaptic structures respond to the loss of primary sensory input. Using in vivo two-photon (2P) imaging, we measured fluctuations in the size of excitatory axonal boutons in L2/3 of adult mouse visual cortex after monocular enucleation. The average size of boutons did not change after deprivation, but the range of bouton sizes was reduced. Large boutons decreased, and small boutons increased. Reduced bouton variance was accompanied by a reduced range of correlated calcium-mediated neural activity in L2/3 of awake animals. Network simulations predicted that size-dependent plasticity may promote conditions of greater bidirectional plasticity. These predictions were supported by electrophysiological measures of short- and long-term plasticity. We propose size-dependent dynamics facilitate cortical reorganization by maximizing the potential for bidirectional plasticity.

In vivo modeling of human neuron dynamics and Down syndrome.

Harnessing the potential of human stem cells for modeling the physiology and diseases of cortical circuitry requires monitoring cellular dynamics in vivo. We show that human induced pluripotent stem cell (iPSC)-derived cortical neurons transplanted into the adult mouse cortex consistently organized into large (up to ~100 mm3) vascularized neuron-glia territories with complex cytoarchitecture. Longitudinal imaging of >4000 grafted developing human neurons revealed that neuronal arbors refined via branch-specific retraction; human synaptic networks substantially restructured over 4 months, with balanced rates of synapse formation and elimination; and oscillatory population activity mirrored the patterns of fetal neural networks. Lastly, we found increased synaptic stability and reduced oscillations in transplants from two individuals with Down syndrome, demonstrating the potential of in vivo imaging in human tissue grafts for patient-specific modeling of cortical development, physiology, and pathogenesis.

Imaging of Brain Slices with a Genetically Encoded Voltage Indicator.

Functional fluorescence microscopy of brain slices using voltage sensitive fluorescent proteins (VSFPs) allows large scale electrophysiological monitoring of neuronal excitation and inhibition. We describe the equipment and techniques needed to successfully record functional responses optical voltage signals from cells expressing a voltage indicator such as VSFP Butterfly 1.2. We also discuss the advantages of voltage imaging and the challenges it presents.