Three-dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval.

Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets, and therefore allows their locations to be pinpointed with precision that achieves the Cramér-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures, from mitochondrial networks and nuclear pores in mammalian cells to amyloid-ß plaques and dendrites in brain tissues and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extracellular targets deep inside tissues.

Spinal Motor Circuit Synaptic Plasticity after Peripheral Nerve Injury Depends on Microglia Activation and a CCR2 Mechanism.

Peripheral nerve injury results in persistent motor deficits, even after the nerve regenerates and muscles are reinnervated. This lack of functional recovery is partly explained by brain and spinal cord circuit alterations triggered by the injury, but the mechanisms are generally unknown. One example of this plasticity is the die-back in the spinal cord ventral horn of the projections of proprioceptive axons mediating the stretch reflex (Ia afferents). Consequently, Ia information about muscle length and dynamics is lost from ventral spinal circuits, degrading motor performance after nerve regeneration. Simultaneously, there is activation of microglia around the central projections of peripherally injured Ia afferents, suggesting a possible causal relationship between neuroinflammation and Ia axon removal. Therefore, we used mice (both sexes) that allow visualization of microglia (CX3CR1-GFP) and infiltrating peripheral myeloid cells (CCR2-RFP) and related changes in these cells to Ia synaptic losses (identified by VGLUT1 content) on retrogradely labeled motoneurons. Microgliosis around axotomized motoneurons starts and peaks within 2 weeks after nerve transection. Thereafter, this region becomes infiltrated by CCR2 cells, and VGLUT1 synapses are lost in parallel. Immunohistochemistry, flow cytometry, and genetic lineage tracing showed that infiltrating CCR2 cells include T cells, dendritic cells, and monocytes, the latter differentiating into tissue macrophages. VGLUT1 synapses were rescued after attenuating the ventral microglial reaction by removal of colony stimulating factor 1 from motoneurons or in CCR2 global KOs. Thus, both activation of ventral microglia and a CCR2-dependent mechanism are necessary for removal of VGLUT1 synapses and alterations in Ia-circuit function following nerve injuries.SIGNIFICANCE STATEMENT Synaptic plasticity and reorganization of essential motor circuits after a peripheral nerve injury can result in permanent motor deficits due to the removal of sensory Ia afferent synapses from the spinal cord ventral horn. Our data link this major circuit change with the neuroinflammatory reaction that occurs inside the spinal cord following injury to peripheral nerves. We describe that both activation of microglia and recruitment into the spinal cord of blood-derived myeloid cells are necessary for motor circuit synaptic plasticity. This study sheds new light into mechanisms that trigger major network plasticity in CNS regions removed from injury sites and that might prevent full recovery of function, even after successful regeneration.

Microglia depletion rapidly and reversibly alters amyloid pathology by modification of plaque compaction and morphologies.

Alzheimer's disease (AD) is a prominent neurodegenerative disorder characterized by deposition of ß-amyloid (Aß)-containing extracellular plaques, accompanied by a microglial-mediated inflammatory response, that leads to cognitive decline. Microglia perform many disease-modifying functions such as phagocytosis of plaques, plaque compaction, and modulation of inflammation through the secretion of cytokines. Microglia are reliant upon colony-stimulating factor receptor-1 (CSF1R) activation for survival. In AD mouse models, chronic targeted depletion of microglia via CSF1R antagonism attenuates plaque formation in early disease but fails to alter plaque burden in late disease. It is unclear if acute depletion of microglia during the peak period of plaque deposition will alter disease pathogenesis, and if so, whether these effects are reversible upon microglial repopulation. To test this, we administered the CSF1R antagonist PLX5622 to the 5XFAD mouse model of AD at four months of age for approximately one month. In a subset of mice, the drug treatment was discontinued, and the mice were fed a control diet for an additional month. We evaluated plaque burden and composition, microgliosis, inflammatory marker expression, and neuritic dystrophy. In 5XFAD animals, CSF1R blockade for 28 days depleted microglia across brain regions by over 50%, suppressed microgliosis, and reduced plaque burden. In microglial-depleted AD animals, neuritic dystrophy was enhanced, and increased diffuse-like plaques and fewer compact-like plaques were observed. Removal of PLX5622 elicited microglial repopulation and subsequent plaque remodeling, resulting in more compact plaques predominating microglia-repopulated regions. We found that microglia limit diffuse plaques by maintaining compact-like plaque properties, thereby blocking the progression of neuritic dystrophy. Microglial repopulation reverses these effects. Collectively, we show that microglia are neuroprotective through maintenance of plaque compaction and morphologies during peak disease progression.

Identification of a novel gene regulating amygdala-mediated fear extinction.

Recent years have seen advances in our understanding of the neural circuits associated with trauma-related disorders, and the development of relevant assays for these behaviors in rodents. Although inherited factors are known to influence individual differences in risk for these disorders, it has been difficult to identify specific genes that moderate circuit functions to affect trauma-related behaviors. Here, we exploited robust inbred mouse strain differences in Pavlovian fear extinction to uncover quantitative trait loci (QTL) associated with this trait. We found these strain differences to be resistant to developmental cross-fostering and associated with anatomical variation in basolateral amygdala (BLA) perineuronal nets, which are developmentally implicated in extinction. Next, by profiling extinction-driven BLA expression of QTL-linked genes, we nominated Ppid (peptidylprolyl isomerase D, a member of the tetratricopeptide repeat (TPR) protein family) as an extinction-related candidate gene. We then showed that Ppid was enriched in excitatory and inhibitory BLA neuronal populations, but at lower levels in the extinction-impaired mouse strain. Using a virus-based approach to directly regulate Ppid function, we demonstrated that downregulating BLA-Ppid impaired extinction, while upregulating BLA-Ppid facilitated extinction and altered in vivo neuronal extinction encoding. Next, we showed that Ppid colocalized with the glucocorticoid receptor (GR) in BLA neurons and found that the extinction-facilitating effects of Ppid upregulation were blocked by a GR antagonist. Collectively, our results identify Ppid as a novel gene involved in regulating extinction via functional actions in the BLA, with possible implications for understanding genetic and pathophysiological mechanisms underlying risk for trauma-related disorders.

Peripheral administration of the soluble TNF inhibitor XPro1595 modifies brain immune cell profiles, decreases beta-amyloid plaque load, and rescues impaired long-term potentiation in 5xFAD mice.

Clinical and animal model studies have implicated inflammation and peripheral immune cell responses in the pathophysiology of Alzheimer's disease (AD). Peripheral immune cells including T cells circulate in the cerebrospinal fluid (CSF) of healthy adults and are found in the brains of AD patients and AD rodent models. Blocking entry of peripheral macrophages into the CNS was reported to increase amyloid burden in an AD mouse model. To assess inflammation in the 5xFAD (Tg) mouse model, we first quantified central and immune cell profiles in the deep cervical lymph nodes and spleen. In the brains of Tg mice, activated (MHCII+, CD45high, and Ly6Chigh) myeloid-derived CD11b+ immune cells are decreased while CD3+ T cells are increased as a function of age relative to non-Tg mice. These immunological changes along with evidence of increased mRNA levels for several cytokines suggest that immune regulation and trafficking patterns are altered in Tg mice. Levels of soluble Tumor Necrosis Factor (sTNF) modulate blood-brain barrier (BBB) permeability and are increased in CSF and brain parenchyma post-mortem in AD subjects and Tg mice. We report here that in vivo peripheral administration of XPro1595, a novel biologic that sequesters sTNF into inactive heterotrimers, reduced the age-dependent increase in activated immune cells in Tg mice, while decreasing the overall number of CD4+ T cells. In addition, XPro1595 treatment in vivo rescued impaired long-term potentiation (LTP) measured in brain slices in association with decreased Aß plaques in the subiculum. Selective targeting of sTNF may modulate brain immune cell infiltration, and prevent or delay neuronal dysfunction in AD. SIGNIFICANCE STATEMENT: Immune cells and cytokines perform specialized functions inside and outside the brain to maintain optimal brain health; but the extent to which their activities change in response to neuronal dysfunction and degeneration is not well understood. Our findings indicate that neutralization of sTNF reduced the age-dependent increase in activated immune cells in Tg mice, while decreasing the overall number of CD4+ T cells. In addition, impaired long-term potentiation (LTP) was rescued by XPro1595 in association with decreased hippocampal Aß plaques. Selective targeting of sTNF holds translational potential to modulate brain immune cell infiltration, dampen neuroinflammation, and prevent or delay neuronal dysfunction in AD.

Soluble TNF mediates amyloid-independent, diet-induced alterations to immune and neuronal functions in an Alzheimer's disease mouse model.

Introduction: Increasing evidence indicates that neurodegenerative diseases, including Alzheimer's disease (AD), are a product of gene-by-environment interplay. The immune system is a major contributor mediating these interactions. Signaling between peripheral immune cells and those within the microvasculature and meninges of the central nervous system (CNS), at the blood-brain barrier, and in the gut likely plays an important role in AD. The cytokine tumor necrosis factor (TNF) is elevated in AD patients, regulates brain and gut barrier permeability, and is produced by central and peripheral immune cells. Our group previously reported that soluble TNF (sTNF) modulates cytokine and chemokine cascades that regulate peripheral immune cell traffic to the brain in young 5xFAD female mice, and in separate studies that a diet high in fat and sugar (HFHS) dysregulates signaling pathways that trigger sTNF-dependent immune and metabolic responses that can result in metabolic syndrome, which is a risk factor for AD. We hypothesized that sTNF is a key mediator of peripheral immune cell contributions to gene-by-environment interactions to AD-like pathology, metabolic dysfunction, and diet-induced gut dysbiosis. Methods: Female 5xFAD mice were subjected to HFHS diet for 2 months and then given XPro1595 to inhibit sTNF for the last month or saline vehicle. We quantified immune cell profiles by multi-color flow cytometry on cells isolated from brain and blood; metabolic, immune, and inflammatory mRNA and protein marker biochemical and immunhistological analyses, gut microbiome, and electrophysiology in brain slices were also performed. Results: Here, we show that selective inhibition of sTNF signaling via the biologic XPro1595 modulates the effects of an HFHS diet in 5xFAD mice on peripheral and central immune profiles including CNS-associated CD8+ T cells, the composition of gut microbiota, and long-term potentiation deficits. Discussion: Obesogenic diet induces immune and neuronal dysfunction in 5xFAD mice and sTNF inhibition mitigates its effects. A clinical trial in subjects at risk for AD due to genetic predisposition and underlying inflammation associated with peripheral inflammatory co-morbidities will be needed to investigate the extent to which these findings translate to the clinic.

Progranulin loss results in sex-dependent dysregulation of the peripheral and central immune system.

Introduction: Progranulin (PGRN) is a secreted glycoprotein, the expression of which is linked to several neurodegenerative diseases. Although its specific function is still unclear, several studies have linked it with lysosomal functions and immune system regulation. Here, we have explored the role of PGRN in peripheral and central immune system homeostasis by investigating the consequences of PGRN deficiency on adaptive and innate immune cell populations. Methods: First, we used gene co-expression network analysis of published data to test the hypothesis that Grn has a critical role in regulating the activation status of immune cell populations in both central and peripheral compartments. To investigate the extent to which PGRN-deficiency resulted in immune dysregulation, we performed deep immunophenotyping by flow cytometry of 19-24-month old male and female Grn-deficient mice (PGRN KO) and littermate Grn-sufficient controls (WT). Results: Male PGRN KO mice exhibited a lower abundance of microglial cells with higher MHC-II expression, increased CD44 expression on monocytes in the brain, and more CNS-associated CD8+ T cells compared to WT mice. Furthermore, we observed an increase in CD44 on CD8+ T cells in the peripheral blood. Female PGRN KO mice also had fewer microglia compared to WT mice, and we also observed reduced expression of MHC-II on brain monocytes. Additionally, we found an increase in Ly-6Chigh monocyte frequency and decreased CD44 expression on CD8+ and CD4+ T cells in PGRN KO female blood. Given that Gpnmb, which encodes for the lysosomal protein Glycoprotein non-metastatic melanoma protein B, has been reported to be upregulated in PGRN KO mice, we investigated changes in GPNMB protein expression associated with PGRN deficits and found that GPNMB is modulated in myeloid cells in a sex-specific manner. Discussion: Our data suggest that PGRN and GPNMB jointly regulate the peripheral and the central immune system in a sex-specific manner; thus, understanding their associated mechanisms could pave the way for developing new neuroprotective strategies to modulate central and peripheral inflammation to lower risk for neurodegenerative diseases and possibly delay or halt progression.

Central amygdala micro-circuits mediate fear extinction.

Fear extinction is an adaptive process whereby defensive responses are attenuated following repeated experience of prior fear-related stimuli without harm. The formation of extinction memories involves interactions between various corticolimbic structures, resulting in reduced central amygdala (CEA) output. Recent studies show, however, the CEA is not merely an output relay of fear responses but contains multiple neuronal subpopulations that interact to calibrate levels of fear responding. Here, by integrating behavioural, in vivo electrophysiological, anatomical and optogenetic approaches in mice we demonstrate that fear extinction produces reversible, stimulus- and context-specific changes in neuronal responses to conditioned stimuli in functionally and genetically defined cell types in the lateral (CEl) and medial (CEm) CEA. Moreover, we show these alterations are absent when extinction is deficient and that selective silencing of protein kinase C delta-expressing (PKCδ) CEl neurons impairs fear extinction. Our findings identify CEA inhibitory microcircuits that act as critical elements within the brain networks mediating fear extinction.

Convergent effects of mouse Pet-1 deletion and human PET-1 variation on amygdala fear and threat processing.

Serotonin is critical for shaping the development of neural circuits regulating emotion. Pet-1 (FEV-1) is an ETS-domain transcription factor essential for differentiation and forebrain targeting of serotonin neurons. Constitutive Pet-1 knockout (KO) causes major loss of serotonin neurons and forebrain serotonin availability, and behavioral abnormalities. We phenotyped Pet-1 KO mice for fear conditioning and extinction, and on a battery of assays for anxiety- and depression-related behaviors. Morphology of Golgi-stained neurons in basolateral amygdala (BLA) and prelimbic cortex was examined. Using human imaging genetics, a common variant (rs860573) in the PET-1 (FEV) gene was tested for effects on threat-related amygdala reactivity and psychopathology in 88 Asian-ancestry subjects. Pet-1 KO mice exhibited increased acquisition and expression of fear, and elevated fear recovery following extinction, relative to wild-type (WT). BLA dendrites of Pet-1 KO mice were significantly longer than in WT. Human PET-1 variation associated with differences in amygdala threat processing and psychopathology. This novel evidence for the role of Pet-1 in fear processing and dendritic organization of amygdala neurons and in human amygdala threat processing extends a growing literature demonstrating the influence of genetic variation in the serotonin system on emotional regulation via effects on structure and function of underlying corticolimbic circuitry.

Genetic strain differences in learned fear inhibition associated with variation in neuroendocrine, autonomic, and amygdala dendritic phenotypes.

Mood and anxiety disorders develop in some but not all individuals following exposure to stress and psychological trauma. However, the factors underlying individual differences in risk and resilience for these disorders, including genetic variation, remain to be determined. Isogenic inbred mouse strains provide a valuable approach to elucidating these factors. Here, we performed a comprehensive examination of the extinction-impaired 129S1/SvImJ (S1) inbred mouse strain for multiple behavioral, autonomic, neuroendocrine, and corticolimbic neuronal morphology phenotypes. We found that S1 exhibited fear overgeneralization to ambiguous contexts and cues, impaired context extinction and impaired safety learning, relative to the (good-extinguishing) C57BL/6J (B6) strain. Fear overgeneralization and impaired extinction was rescued by treatment with the front-line anxiety medication fluoxetine. Telemetric measurement of electrocardiogram signals demonstrated autonomic disturbances in S1 including poor recovery of fear-induced suppression of heart rate variability. S1 with a history of chronic restraint stress displayed an attenuated corticosterone (CORT) response to a novel, swim stressor. Conversely, previously stress-naive S1 showed exaggerated CORT responses to acute restraint stress or extinction training, insensitivity to dexamethasone challenge, and reduced hippocampal CA3 glucocorticoid receptor mRNA, suggesting downregulation of negative feedback control of the hypothalamic-pituitary-adrenal axis. Analysis of neuronal morphology in key neural nodes within the fear and extinction circuit revealed enlarged dendritic arbors in basolateral amygdala neurons in S1, but normal infralimbic cortex and prelimbic cortex dendritic arborization. Collectively, these data provide convergent support for the utility of the S1 strain as a tractable model for elucidating the neural, molecular and genetic basis of persistent, excessive fear.

Chronic alcohol remodels prefrontal neurons and disrupts NMDAR-mediated fear extinction encoding.

Alcoholism is frequently co-morbid with post-traumatic stress disorder, but it is unclear how alcohol affects the neural circuits mediating recovery from trauma. We found that chronic intermittent ethanol (CIE) impaired fear extinction and remodeled the dendritic arbor of medial prefrontal cortical (mPFC) neurons in mice. CIE impaired extinction encoding by infralimbic mPFC neurons in vivo and functionally downregulated burst-mediating NMDA GluN1 receptors. These findings suggest that alcohol may increase risk for trauma-related anxiety disorders by disrupting mPFC-mediated extinction of fear.