Publications by authors named "Jaime Grutzendler"

50 Publications

Intravital Imaging of Neocortical Heterotopia Reveals Aberrant Axonal Pathfinding and Myelination around Ectopic Neurons.

Cereb Cortex 2021 Apr 20. Epub 2021 Apr 20.

Interdepartmental Neuroscience Program, Yale School of Medicine, New Haven, CT 06510, USA.

Neocortical heterotopia consist of ectopic neuronal clusters that are frequently found in individuals with cognitive disability and epilepsy. However, their pathogenesis remains poorly understood due in part to a lack of tractable animal models. We have developed an inducible model of focal cortical heterotopia that enables their precise spatiotemporal control and high-resolution optical imaging in live mice. Here, we report that heterotopia are associated with striking patterns of circumferentially projecting axons and increased myelination around neuronal clusters. Despite their aberrant axonal patterns, in vivo calcium imaging revealed that heterotopic neurons remain functionally connected to other brain regions, highlighting their potential to influence global neural networks. These aberrant patterns only form when heterotopia are induced during a critical embryonic temporal window, but not in early postnatal development. Our model provides a new way to investigate heterotopia formation in vivo and reveals features suggesting the existence of developmentally modulated, neuron-derived axon guidance and myelination factors.
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http://dx.doi.org/10.1093/cercor/bhab090DOI Listing
April 2021

Unlocking Pericyte Function in the Adult Blood Brain Barrier One Cell at a Time.

Circ Res 2021 Feb 18;128(4):511-512. Epub 2021 Feb 18.

Neurology (J.G.), Yale University School of Medicine, New Haven, CT.

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http://dx.doi.org/10.1161/CIRCRESAHA.121.318799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928238PMC
February 2021

Caveolae-mediated Tie2 signaling contributes to CCM pathogenesis in a brain endothelial cell-specific Pdcd10-deficient mouse model.

Nat Commun 2021 01 25;12(1):504. Epub 2021 Jan 25.

Interdepartmental Program in Vascular Biology and Therapeutics, Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.

Cerebral cavernous malformations (CCMs) are vascular abnormalities that primarily occur in adulthood and cause cerebral hemorrhage, stroke, and seizures. CCMs are thought to be initiated by endothelial cell (EC) loss of any one of the three Ccm genes: CCM1 (KRIT1), CCM2 (OSM), or CCM3 (PDCD10). Here we report that mice with a brain EC-specific deletion of Pdcd10 (Pdcd10) survive up to 6-12 months and develop bona fide CCM lesions in all regions of brain, allowing us to visualize the vascular dynamics of CCM lesions using transcranial two-photon microscopy. This approach reveals that CCMs initiate from protrusion at the level of capillary and post-capillary venules with gradual dissociation of pericytes. Microvascular beds in lesions are hyper-permeable, and these disorganized structures present endomucin-positive ECs and α-smooth muscle actin-positive pericytes. Caveolae in the endothelium of Pdcd10 lesions are drastically increased, enhancing Tie2 signaling in Ccm3-deficient ECs. Moreover, genetic deletion of caveolin-1 or pharmacological blockade of Tie2 signaling effectively normalizes microvascular structure and barrier function with attenuated EC-pericyte disassociation and CCM lesion formation in Pdcd10 mice. Our study establishes a chronic CCM model and uncovers a mechanism by which CCM3 mutation-induced caveolae-Tie2 signaling contributes to CCM pathogenesis.
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http://dx.doi.org/10.1038/s41467-020-20774-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835246PMC
January 2021

Imaging and optogenetic modulation of vascular mural cells in the live brain.

Nat Protoc 2021 01 9;16(1):472-496. Epub 2020 Dec 9.

Department of Neurology, Yale School of Medicine, New Haven, CT, USA.

Mural cells (smooth muscle cells and pericytes) are integral components of brain blood vessels that play important roles in vascular formation, blood-brain barrier maintenance, and regulation of regional cerebral blood flow (rCBF). These cells are implicated in conditions ranging from developmental vascular disorders to age-related neurodegenerative diseases. Here we present complementary tools for cell labeling with transgenic mice and organic dyes that allow high-resolution intravital imaging of the different mural cell subtypes. We also provide detailed methodologies for imaging of spontaneous and neural activity-evoked calcium transients in mural cells. In addition, we describe strategies for single- and two-photon optogenetics that allow manipulation of the activity of individual and small clusters of mural cells. Together with measurements of diameter and flow in individual brain microvessels, calcium imaging and optogenetics allow the investigation of pericyte and smooth muscle cell physiology and their role in regulating rCBF. We also demonstrate the utility of these tools to investigate mural cells in the context of Alzheimer's disease and cerebral ischemia mouse models. Thus, these methods can be used to reveal the functional and structural heterogeneity of mural cells in vivo, and allow detailed cellular studies of the normal function and pathophysiology of mural cells in a variety of disease models. The implementation of this protocol can take from several hours to days depending on the intended applications.
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http://dx.doi.org/10.1038/s41596-020-00425-wDOI Listing
January 2021

Astrocytes and microglia play orchestrated roles and respect phagocytic territories during neuronal corpse removal in vivo.

Sci Adv 2020 Jun 26;6(26):eaba3239. Epub 2020 Jun 26.

Department of Neurology, Yale School of Medicine, New Haven, CT, USA.

Cell death is prevalent throughout life; however, the coordinated interactions and roles of phagocytes during corpse removal in the live brain are poorly understood. We developed photochemical and viral methodologies to induce death in single cells and combined this with intravital optical imaging. This approach allowed us to track multicellular phagocytic interactions with precise spatiotemporal resolution. Astrocytes and microglia engaged with dying neurons in an orchestrated and synchronized fashion. Each glial cell played specialized roles: Astrocyte processes rapidly polarized and engulfed numerous small dendritic apoptotic bodies, while microglia migrated and engulfed the soma and apical dendrites. The relative involvement and phagocytic specialization of each glial cell was plastic and controlled by the receptor tyrosine kinase . In aging, there was a marked delay in apoptotic cell removal. Thus, a precisely orchestrated response and cross-talk between glial cells during corpse removal may be critical for maintaining brain homeostasis.
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http://dx.doi.org/10.1126/sciadv.aba3239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319765PMC
June 2020

TREM2: Modulator of Lipid Metabolism in Microglia.

Neuron 2020 03;105(5):759-761

Department of Neurology, Yale School of Medicine, New Haven, CT, USA; Department of Neuroscience, Yale School of Medicine, New Haven, CT, USA. Electronic address:

Lipid-processing mechanisms during demyelination are poorly understood. In this issue of Neuron,Nugent et al. (2020) show by cell-specific lipidomics that Trem2 deficiency leads to cholesterol ester (CE) overload in microglia. This is mediated by misregulation of lipid metabolism genes and is rescued by modulating CE synthesis or efflux.
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http://dx.doi.org/10.1016/j.neuron.2020.02.008DOI Listing
March 2020

Emerging technologies to study glial cells.

Glia 2020 09 20;68(9):1692-1728. Epub 2020 Jan 20.

Commissariat à l'Energie Atomique et aux Energies Alternatives, Département de la Recherche Fondamentale, Institut de Biologie François Jacob, MIRCen, Fontenay-aux-Roses, France.

Development, physiological functions, and pathologies of the brain depend on tight interactions between neurons and different types of glial cells, such as astrocytes, microglia, oligodendrocytes, and oligodendrocyte precursor cells. Assessing the relative contribution of different glial cell types is required for the full understanding of brain function and dysfunction. Over the recent years, several technological breakthroughs were achieved, allowing "glio-scientists" to address new challenging biological questions. These technical developments make it possible to study the roles of specific cell types with medium or high-content workflows and perform fine analysis of their mutual interactions in a preserved environment. This review illustrates the potency of several cutting-edge experimental approaches (advanced cell cultures, induced pluripotent stem cell (iPSC)-derived human glial cells, viral vectors, in situ glia imaging, opto- and chemogenetic approaches, and high-content molecular analysis) to unravel the role of glial cells in specific brain functions or diseases. It also illustrates the translation of some techniques to the clinics, to monitor glial cells in patients, through specific brain imaging methods. The advantages, pitfalls, and future developments are discussed for each technique, and selected examples are provided to illustrate how specific "gliobiological" questions can now be tackled.
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http://dx.doi.org/10.1002/glia.23780DOI Listing
September 2020

Cellular Control of Brain Capillary Blood Flow: In Vivo Imaging Veritas.

Trends Neurosci 2019 08 26;42(8):528-536. Epub 2019 Jun 26.

Center for Translational Neuromedicine, University of Rochester Medical Center, Elmwood Avenue 601, Rochester, NY 14642, USA; Center for Translational Neuromedicine, Faculty of Medical and Health Sciences, University of Copenhagen, Denmark, Blegdamsvej 3B, 2200 Copenhagen N, Denmark. Electronic address:

The precise modulation of regional cerebral blood flow during neural activation is important for matching local energetic demand and supply and clearing brain metabolites. Here we discuss advances facilitated by high-resolution optical in vivo imaging techniques that for the first time have provided direct visualization of capillary blood flow and its modulation by neural activity. We focus primarily on studies of microvascular flow, mural cell control of vessel diameter, and oxygen level-dependent changes in red blood cell deformability. We also suggest methodological standards for best practices when studying microvascular perfusion, partly motivated by recent controversies about the precise location within the microvascular tree where neurovascular coupling is initiated, and the role of mural cells in the control of vasomotility.
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http://dx.doi.org/10.1016/j.tins.2019.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386067PMC
August 2019

Uncovering the biology of myelin with optical imaging of the live brain.

Glia 2019 11 29;67(11):2008-2019. Epub 2019 Apr 29.

Departments of Neurology and Neuroscience, Yale School of Medicine, New Haven, Connecticut.

Myelin has traditionally been considered a static structure that is produced and assembled during early developmental stages. While this characterization is accurate in some contexts, recent studies have revealed that oligodendrocyte generation and patterns of myelination are dynamic and potentially modifiable throughout life. Unique structural and biochemical properties of the myelin sheath provide opportunities for the development and implementation of multimodal label-free and fluorescence optical imaging approaches. When combined with genetically encoded fluorescent tags targeted to distinct cells and subcellular structures, these techniques offer a powerful methodological toolbox for uncovering mechanisms of myelin generation and plasticity in the live brain. Here, we discuss recent advances in these approaches that have allowed the discovery of several forms of myelin plasticity in developing and adult nervous systems. Using these techniques, long-standing questions related to myelin generation, remodeling, and degeneration can now be addressed.
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http://dx.doi.org/10.1002/glia.23635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744352PMC
November 2019

Publisher Correction: Flexible Learning-Free Segmentation and Reconstruction of Neural Volumes.

Sci Rep 2018 Nov 29;8(1):17585. Epub 2018 Nov 29.

Department of Computer Science and Engineering, University of Notre Dame, Notre Dame, IN, USA.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-36220-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265300PMC
November 2018

Flexible Learning-Free Segmentation and Reconstruction of Neural Volumes.

Sci Rep 2018 09 24;8(1):14247. Epub 2018 Sep 24.

Department of Computer Science and Engineering, University of Notre Dame, Notre Dame, IN, USA.

Imaging is a dominant strategy for data collection in neuroscience, yielding stacks of images that often scale to gigabytes of data for a single experiment. Machine learning algorithms from computer vision can serve as a pair of virtual eyes that tirelessly processes these images, automatically detecting and identifying microstructures. Unlike learning methods, our Flexible Learning-free Reconstruction of Imaged Neural volumes (FLoRIN) pipeline exploits structure-specific contextual clues and requires no training. This approach generalizes across different modalities, including serially-sectioned scanning electron microscopy (sSEM) of genetically labeled and contrast enhanced processes, spectral confocal reflectance (SCoRe) microscopy, and high-energy synchrotron X-ray microtomography (μCT) of large tissue volumes. We deploy the FLoRIN pipeline on newly published and novel mouse datasets, demonstrating the high biological fidelity of the pipeline's reconstructions. FLoRIN reconstructions are of sufficient quality for preliminary biological study, for example examining the distribution and morphology of cells or extracting single axons from functional data. Compared to existing supervised learning methods, FLoRIN is one to two orders of magnitude faster and produces high-quality reconstructions that are tolerant to noise and artifacts, as is shown qualitatively and quantitatively.
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http://dx.doi.org/10.1038/s41598-018-32628-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155135PMC
September 2018

Lifelong cortical myelin plasticity and age-related degeneration in the live mammalian brain.

Nat Neurosci 2018 05 19;21(5):683-695. Epub 2018 Mar 19.

Department of Neurology, Yale School of Medicine, New Haven, CT, USA.

Axonal myelin increases neural processing speed and efficiency. It is unknown whether patterns of myelin distribution are fixed or whether myelinating oligodendrocytes are continually generated in adulthood and maintain the capacity for structural remodeling. Using high-resolution, intravital label-free and fluorescence optical imaging in mouse cortex, we demonstrate lifelong oligodendrocyte generation occurring in parallel with structural plasticity of individual myelin internodes. Continuous internode formation occurred on both partially myelinated and unmyelinated axons, and the total myelin coverage along individual axons progressed up to two years of age. After peak myelination, gradual oligodendrocyte death and myelin degeneration in aging were associated with pronounced internode loss and myelin debris accumulation within microglia. Thus, cortical myelin remodeling is protracted throughout life, potentially playing critical roles in neuronal network homeostasis. The gradual loss of internodes and myelin degeneration in aging could contribute significantly to brain pathogenesis.
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http://dx.doi.org/10.1038/s41593-018-0120-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920745PMC
May 2018

Activation of pial and dural macrophages and dendritic cells by cortical spreading depression.

Ann Neurol 2018 03 13;83(3):508-521. Epub 2018 Mar 13.

Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Boston, MA.

Objective: Cortical spreading depression (CSD) has long been implicated in migraine attacks with aura. The process by which CSD, a cortical event that occurs within the blood-brain barrier (BBB), results in nociceptor activation outside the BBB is likely mediated by multiple molecules and cells. The objective of this study was to determine whether CSD activates immune cells inside the BBB (pia), outside the BBB (dura), or in both, and if so, when.

Methods: Investigating cellular events in the meninges shortly after CSD, we used in vivo two-photon imaging to identify changes in macrophages and dendritic cells (DCs) that reside in the pia, arachnoid, and dura and their anatomical relationship to TRPV1 axons.

Results: We found that activated meningeal macrophages retract their processes and become circular, and that activated meningeal DCs stop migrating. We found that CSD activates pial macrophages instantaneously, pial, subarachnoid, and dural DCs 6-12 minutes later, and dural macrophages 20 minutes later. Dural macrophages and DCs can appear in close proximity to TRPV1-positive axons.

Interpretation: The findings suggest that activation of pial macrophages may be more relevant to cases where aura and migraine begin simultaneously, that activation of dural macrophages may be more relevant to cases where headache begins 20 to 30 minutes after aura, and that activation of dural macrophages may be mediated by activation of migratory DCs in the subarachnoid space and dura. The anatomical relationship between TRPV1-positive meningeal nociceptors, and dural macrophages and DCs supports a role for these immune cells in the modulation of head pain. Ann Neurol 2018;83:508-521.
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http://dx.doi.org/10.1002/ana.25169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965700PMC
March 2018

Microglia-Mediated Neuroprotection, TREM2, and Alzheimer's Disease: Evidence From Optical Imaging.

Biol Psychiatry 2018 02 14;83(4):377-387. Epub 2017 Oct 14.

Department of Neurology, Yale School of Medicine, New Haven, Connecticut; Department of Neuroscience, Yale School of Medicine, New Haven, Connecticut. Electronic address:

Recent genetic studies have provided overwhelming evidence of the involvement of microglia-related molecular networks in the pathophysiology of Alzheimer's disease (AD). However, the precise mechanisms by which microglia alter the course of AD neuropathology remain poorly understood. Here we discuss current evidence of the neuroprotective functions of microglia with a focus on optical imaging studies that have revealed a role of these cells in the encapsulation of amyloid deposits ("microglia barrier"). This barrier modulates the degree of plaque compaction, amyloid fibril surface area, and insulation from adjacent axons thereby reducing neurotoxicity. We discuss findings implicating genetic variants of the microglia receptor, triggering receptor expressed on myeloid cells 2, in the increased risk of late onset AD. We provide evidence that increased AD risk may be at least partly mediated by deficient microglia polarization toward amyloid deposits, resulting in ineffective plaque encapsulation and reduced plaque compaction, which is associated with worsened axonal pathology. Finally, we propose possible avenues for therapeutic targeting of plaque-associated microglia with the goal of enhancing the microglia barrier and potentially reducing disease progression.
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http://dx.doi.org/10.1016/j.biopsych.2017.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767550PMC
February 2018

Oxalate-curcumin-based probe for micro- and macroimaging of reactive oxygen species in Alzheimer's disease.

Proc Natl Acad Sci U S A 2017 11 6;114(47):12384-12389. Epub 2017 Nov 6.

Molecular Imaging Laboratory, Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129;

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that has a progression that is closely associated with oxidative stress. It has long been speculated that the reactive oxygen species (ROS) level in AD brains is much higher than that in healthy brains. However, evidence from living beings is scarce. Inspired by the "chemistry of glow stick," we designed a near-IR fluorescence (NIRF) imaging probe, termed CRANAD-61, for sensing ROS to provide evidence at micro- and macrolevels. In CRANAD-61, an oxalate moiety was utilized to react with ROS and to consequentially produce wavelength shifting. Our in vitro data showed that CRANAD-61 was highly sensitive and rapidly responsive to various ROS. On reacting with ROS, its excitation and emission wavelengths significantly shifted to short wavelengths, and this shifting could be harnessed for dual-color two-photon imaging and transformative NIRF imaging. In this report, we showed that CRANAD-61 could be used to identify "active" amyloid beta (Aβ) plaques and cerebral amyloid angiopathy (CAA) surrounded by high ROS levels with two-photon imaging (microlevel) and to provide relative total ROS concentrations in AD brains via whole-brain NIRF imaging (macrolevel). Lastly, we showed that age-related increases in ROS levels in AD brains could be monitored with our NIRF imaging method. We believe that our imaging with CRANAD-61 could provide evidence of ROS at micro- and macrolevels and could be used for monitoring ROS changes under various AD pathological conditions and during drug treatment.
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http://dx.doi.org/10.1073/pnas.1706248114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703278PMC
November 2017

Targeted two-photon chemical apoptotic ablation of defined cell types in vivo.

Nat Commun 2017 06 16;8:15837. Epub 2017 Jun 16.

Department of Neurology, Yale School of Medicine, New Haven, Connecticut 06511, USA.

A major bottleneck limiting understanding of mechanisms and consequences of cell death in complex organisms is the inability to induce and visualize this process with spatial and temporal precision in living animals. Here we report a technique termed two-photon chemical apoptotic targeted ablation (2Phatal) that uses focal illumination with a femtosecond-pulsed laser to bleach a nucleic acid-binding dye causing dose-dependent apoptosis of individual cells without collateral damage. Using 2Phatal, we achieve precise ablation of distinct populations of neurons, glia and pericytes in the mouse brain and in zebrafish. When combined with organelle-targeted fluorescent proteins and biosensors, we uncover previously unrecognized cell-type differences in patterns of apoptosis and associated dynamics of ribosomal disassembly, calcium overload and mitochondrial fission. 2Phatal provides a powerful and rapidly adoptable platform to investigate in vivo functional consequences and neural plasticity following cell death as well as apoptosis, cell clearance and tissue remodelling in diverse organs and species.
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http://dx.doi.org/10.1038/ncomms15837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501159PMC
June 2017

A fluoro-Nissl dye identifies pericytes as distinct vascular mural cells during in vivo brain imaging.

Nat Neurosci 2017 Jul 15;20(7):1023-1032. Epub 2017 May 15.

Department of Neurology, Yale School of Medicine, New Haven, Connecticut, USA.

Pericytes and smooth muscle cells are integral components of the brain microvasculature. However, no techniques exist to unambiguously identify these cell types, greatly limiting their investigation in vivo. Here we show that the fluorescent Nissl dye NeuroTrace 500/525 labels brain pericytes with specificity, allowing high-resolution optical imaging in the live mouse. We demonstrate that capillary pericytes are a population of mural cells with distinct morphological, molecular and functional features that do not overlap with precapillary or arteriolar smooth muscle actin-expressing cells. The remarkable specificity for dye uptake suggests that pericytes have molecular transport mechanisms not present in other brain cells. We demonstrate feasibility of longitudinal pericyte imaging during microvascular development and aging and in models of brain ischemia and Alzheimer's disease. The ability to easily label pericytes in any mouse model opens the possibility of a broad range of investigations of mural cells in vascular development, neurovascular coupling and neuropathology.
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http://dx.doi.org/10.1038/nn.4564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550770PMC
July 2017

TREM2 Haplodeficiency in Mice and Humans Impairs the Microglia Barrier Function Leading to Decreased Amyloid Compaction and Severe Axonal Dystrophy.

Neuron 2016 05;90(4):724-39

Department of Neurology, Yale University, New Haven, CT 06511, USA; Department of Neuroscience, Yale University, New Haven, CT 06511, USA. Electronic address:

Haplodeficiency of the microglia gene TREM2 increases risk for late-onset Alzheimer's disease (AD) but the mechanisms remain uncertain. To investigate this, we used high-resolution confocal and super-resolution (STORM) microscopy in AD-like mice and human AD tissue. We found that microglia processes, rich in TREM2, tightly surround early amyloid fibrils and plaques promoting their compaction and insulation. In Trem2- or DAP12-haplodeficient mice and in humans with R47H TREM2 mutations, microglia had a markedly reduced ability to envelop amyloid deposits. This led to an increase in less compact plaques with longer and branched amyloid fibrils resulting in greater surface exposure to adjacent neurites. This was associated with more severe neuritic tau hyperphosphorylation and axonal dystrophy around amyloid deposits. Thus, TREM2 deficiency may disrupt the formation of a neuroprotective microglia barrier that regulates amyloid compaction and insulation. Pharmacological modulation of this barrier could be a novel therapeutic strategy for AD.
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http://dx.doi.org/10.1016/j.neuron.2016.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4898967PMC
May 2016

TREM2-mediated early microglial response limits diffusion and toxicity of amyloid plaques.

J Exp Med 2016 05 18;213(5):667-75. Epub 2016 Apr 18.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial receptor that recognizes changes in the lipid microenvironment, which may occur during amyloid β (Aβ) accumulation and neuronal degeneration in Alzheimer's disease (AD). Rare TREM2 variants that affect TREM2 function lead to an increased risk of developing AD. In murine models of AD, TREM2 deficiency prevents microglial clustering around Aβ deposits. However, the origin of myeloid cells surrounding amyloid and the impact of TREM2 on Aβ accumulation are a matter of debate. Using parabiosis, we found that amyloid-associated myeloid cells derive from brain-resident microglia rather than from recruitment of peripheral blood monocytes. To determine the impact of TREM2 deficiency on Aβ accumulation, we examined Aβ plaques in the 5XFAD model of AD at the onset of Aβ-related pathology. At this early time point, Aβ accumulation was similar in TREM2-deficient and -sufficient 5XFAD mice. However, in the absence of TREM2, Aβ plaques were not fully enclosed by microglia; they were more diffuse, less dense, and were associated with significantly greater neuritic damage. Thus, TREM2 protects from AD by enabling microglia to surround and alter Aβ plaque structure, thereby limiting neuritic damage.
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http://dx.doi.org/10.1084/jem.20151948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854736PMC
May 2016

Increased Nanoparticle Delivery to Brain Tumors by Autocatalytic Priming for Improved Treatment and Imaging.

ACS Nano 2016 04 16;10(4):4209-18. Epub 2016 Mar 16.

Department of Neurosurgery, #Department of Biomedical Engineering, ‡PET Center, Department of Diagnostic Radiology, §Department of Neurology, and ⊥Department of Pathology, Yale University , New Haven, Connecticut 06511, United States.

The blood-brain barrier (BBB) is partially disrupted in brain tumors. Despite the gaps in the BBB, there is an inadequate amount of pharmacological agents delivered into the brain. Thus, the low delivery efficiency renders many of these agents ineffective in treating brain cancer. In this report, we proposed an "autocatalytic" approach for increasing the transport of nanoparticles into the brain. In this strategy, a small number of nanoparticles enter into the brain via transcytosis or through the BBB gaps. After penetrating the BBB, the nanoparticles release BBB modulators, which enables more nanoparticles to be transported, creating a positive feedback loop for increased delivery. Specifically, we demonstrated that these autocatalytic brain tumor-targeting poly(amine-co-ester) terpolymer nanoparticles (ABTT NPs) can readily cross the BBB and preferentially accumulate in brain tumors at a concentration of 4.3- and 94.0-fold greater than that in the liver and in brain regions without tumors, respectively. We further demonstrated that ABTT NPs were capable of mediating brain cancer gene therapy and chemotherapy. Our results suggest ABTT NPs can prime the brain to increase the systemic delivery of therapeutics for treating brain malignancies.
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http://dx.doi.org/10.1021/acsnano.5b07573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5257033PMC
April 2016

Neurovascular and Immuno-Imaging: From Mechanisms to Therapies. Proceedings of the Inaugural Symposium.

Front Neurosci 2016 22;10:46. Epub 2016 Feb 22.

Department of Physiology and Biophysics, Keck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California Los Angeles, CA, USA.

Breakthrough advances in intravital imaging have launched a new era for the study of dynamic interactions at the neurovascular interface in health and disease. The first Neurovascular and Immuno-Imaging Symposium was held at the Gladstone Institutes, University of California, San Francisco in March, 2015. This highly interactive symposium brought together a group of leading researchers who discussed how recent studies have unraveled fundamental biological mechanisms in diverse scientific fields such as neuroscience, immunology, and vascular biology, both under physiological and pathological conditions. These Proceedings highlight how advances in imaging technologies and their applications revolutionized our understanding of the communication between brain, immune, and vascular systems and identified novel targets for therapeutic intervention in neurological diseases.
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http://dx.doi.org/10.3389/fnins.2016.00046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4761864PMC
March 2016

Attenuation of β-Amyloid Deposition and Neurotoxicity by Chemogenetic Modulation of Neural Activity.

J Neurosci 2016 Jan;36(2):632-41

Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut 06510

Unlabelled: Aberrant neural hyperactivity has been observed in early stages of Alzheimer's disease (AD) and may be a driving force in the progression of amyloid pathology. Evidence for this includes the findings that neural activity may modulate β-amyloid (Aβ) peptide secretion and experimental stimulation of neural activity can increase amyloid deposition. However, whether long-term attenuation of neural activity prevents the buildup of amyloid plaques and associated neural pathologies remains unknown. Using viral-mediated delivery of designer receptors exclusively activated by designer drugs (DREADDs), we show in two AD-like mouse models that chronic intermittent increases or reductions of activity have opposite effects on Aβ deposition. Neural activity reduction markedly decreases Aβ aggregation in regions containing axons or dendrites of DREADD-expressing neurons, suggesting the involvement of synaptic and nonsynaptic Aβ release mechanisms. Importantly, activity attenuation is associated with a reduction in axonal dystrophy and synaptic loss around amyloid plaques. Thus, modulation of neural activity could constitute a potential therapeutic strategy for ameliorating amyloid-induced pathology in AD.

Significance Statement: A novel chemogenetic approach to upregulate and downregulate neuronal activity in Alzheimer's disease (AD) mice was implemented. This led to the first demonstration that chronic intermittent attenuation of neuronal activity in vivo significantly reduces amyloid deposition. The study also demonstrates that modulation of β-amyloid (Aβ) release can occur at both axonal and dendritic fields, suggesting the involvement of synaptic and nonsynaptic Aβ release mechanisms. Activity reductions also led to attenuation of the synaptic pathology associated with amyloid plaques. Therefore, chronic attenuation of neuronal activity could constitute a novel therapeutic approach for AD.
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http://dx.doi.org/10.1523/JNEUROSCI.2531-15.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710779PMC
January 2016

Absolute two-photon excitation spectra of red and far-red fluorescent probes.

Opt Lett 2015 Nov;40(21):4915-8

Efficient use of two-photon excitation (TPE) microscopy requires knowledge of the absolute TPE action cross sections (ATACSs) of fluorescent probes. However, these values are not available for recently developed dyes, which exhibit superior properties in many modern microscopy applications. We report ATACSs of five red to far-red organic dyes, ATTO 647N, STAR 635P, silicon rhodamine, ATTO 594, and ATTO 590. The dyes were found to have large ATACSs (>100  GM) at their respective wavelength peaks, thus supporting their use as bright fluorescent markers in TPE microscopy.
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http://dx.doi.org/10.1364/OL.40.004915DOI Listing
November 2015

Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques.

Proc Natl Acad Sci U S A 2015 Jul 29;112(28):E3699-708. Epub 2015 Jun 29.

Department of Cell Biology, Program in Cellular Neuroscience, Neurodegeneration and Repair,

Through a comprehensive analysis of organellar markers in mouse models of Alzheimer's disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer's disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer's disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology.
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http://dx.doi.org/10.1073/pnas.1510329112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507205PMC
July 2015

Regional Blood Flow in the Normal and Ischemic Brain Is Controlled by Arteriolar Smooth Muscle Cell Contractility and Not by Capillary Pericytes.

Neuron 2015 Jul 25;87(1):95-110. Epub 2015 Jun 25.

Department of Neurology, Yale School of Medicine, New Haven, CT 06510, USA; Department of Neurobiology, Yale School of Medicine, New Haven, CT 06510, USA. Electronic address:

The precise regulation of cerebral blood flow is critical for normal brain function, and its disruption underlies many neuropathologies. The extent to which smooth muscle-covered arterioles or pericyte-covered capillaries control vasomotion during neurovascular coupling remains controversial. We found that capillary pericytes in mice and humans do not express smooth muscle actin and are morphologically and functionally distinct from adjacent precapillary smooth muscle cells (SMCs). Using optical imaging we investigated blood flow regulation at various sites on the vascular tree in living mice. Optogenetic, whisker stimulation, or cortical spreading depolarization caused microvascular diameter or flow changes in SMC but not pericyte-covered microvessels. During early stages of brain ischemia, transient SMC but not pericyte constrictions were a major cause of hypoperfusion leading to thrombosis and distal microvascular occlusions. Thus, capillary pericytes are not contractile, and regulation of cerebral blood flow in physiological and pathological conditions is mediated by arteriolar SMCs.
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http://dx.doi.org/10.1016/j.neuron.2015.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487786PMC
July 2015

Genetic variants associated with autoimmunity drive NFκB signaling and responses to inflammatory stimuli.

Sci Transl Med 2015 Jun;7(291):291ra93

Department of Neurology and Immunobiology, Yale School of Medicine, New Haven, CT 06511, USA. Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, MA 02142, USA.

The transcription factor nuclear factor κB (NFκB) is a central regulator of inflammation, and genome-wide association studies in subjects with autoimmune disease have identified a number of variants within the NFκB signaling cascade. In addition, causal variant fine-mapping has demonstrated that autoimmune disease susceptibility variants for multiple sclerosis (MS) and ulcerative colitis are strongly enriched within binding sites for NFκB. We report that MS-associated variants proximal to NFκB1 and in an intron of TNFRSF1A (TNFR1) are associated with increased NFκB signaling after tumor necrosis factor-α (TNFα) stimulation. Both variants result in increased degradation of inhibitor of NFκB α (IκBα), a negative regulator of NFκB, and nuclear translocation of p65 NFκB. The variant proximal to NFκB1 controls signaling responses by altering the expression of NFκB itself, with the GG risk genotype expressing 20-fold more p50 NFκB and diminished expression of the negative regulators of the NFκB pathway: TNFα-induced protein 3 (TNFAIP3), B cell leukemia 3 (BCL3), and cellular inhibitor of apoptosis 1 (CIAP1). Finally, naïve CD4 T cells from patients with MS express enhanced activation of p65 NFκB. These results demonstrate that genetic variants associated with risk of developing MS alter NFκB signaling pathways, resulting in enhanced NFκB activation and greater responsiveness to inflammatory stimuli. As such, this suggests that rapid genetic screening for variants associated with NFκB signaling may identify individuals amenable to NFκB or cytokine blockade.
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http://dx.doi.org/10.1126/scitranslmed.aaa9223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574294PMC
June 2015

Microglia constitute a barrier that prevents neurotoxic protofibrillar Aβ42 hotspots around plaques.

Nat Commun 2015 Jan 29;6:6176. Epub 2015 Jan 29.

1] Department of Neurology, Yale University, New Haven, Connecticut 06511, USA [2] Department of Neurobiology, Yale University, New Haven, Connecticut 06510, USA.

In Alzheimer's disease (AD), β-amyloid (Aβ) plaques are tightly enveloped by microglia processes, but the significance of this phenomenon is unknown. Here we show that microglia constitute a barrier with profound impact on plaque composition and toxicity. Using high-resolution confocal and in vivo two-photon imaging in AD mouse models, we demonstrate that this barrier prevents outward plaque expansion and leads to compact plaque microregions with low Aβ42 affinity. Areas uncovered by microglia are less compact but have high Aβ42 affinity, leading to the formation of protofibrillar Aβ42 hotspots that are associated with more severe axonal dystrophy. In ageing, microglia coverage is reduced leading to enlarged protofibrillar Aβ42 hotspots and more severe neuritic dystrophy. CX3CR1 gene deletion or anti-Aβ immunotherapy causes expansion of microglia coverage and reduced neuritic dystrophy. Failure of the microglia barrier and the accumulation of neurotoxic protofibrillar Aβ hotspots may constitute novel therapeutic and clinical imaging targets for AD.
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http://dx.doi.org/10.1038/ncomms7176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311408PMC
January 2015

In vivo imaging of oligodendrocytes with sulforhodamine 101.

Nat Methods 2014 Nov;11(11):1081-2

1] Department of Neurology, Yale School of Medicine, New Haven, Connecticut, USA. [2] Department of Neurobiology, Yale School of Medicine, New Haven, Connecticut, USA.

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http://dx.doi.org/10.1038/nmeth.3140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539948PMC
November 2014

Modulation of oligodendrocyte generation during a critical temporal window after NG2 cell division.

Nat Neurosci 2014 Nov 28;17(11):1518-27. Epub 2014 Sep 28.

1] Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut, USA. [2] Connecticut Stem Cell Institute, University of Connecticut, Farmington, Connecticut, USA.

Oligodendrocytes in the mammalian brain are continuously generated from NG2 cells throughout postnatal life. However, it is unclear when the decision is made for NG2 cells to self-renew or differentiate into oligodendrocytes after cell division. Using a combination of in vivo and ex vivo imaging and fate analysis of proliferated NG2 cells in fixed tissue, we demonstrate that in the postnatal developing mouse brain, the majority of divided NG2 cells differentiate into oligodendrocytes during a critical age-specific temporal window of 3-8 d. Notably, within this time period, damage to myelin and oligodendrocytes accelerated oligodendrocyte differentiation from divided cells, and whisker removal decreased the survival of divided cells in the deprived somatosensory cortex. These findings indicate that during the critical temporal window of plasticity, the fate of divided NG2 cells is sensitive to modulation by external signals.
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http://dx.doi.org/10.1038/nn.3815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275302PMC
November 2014