High-resolution vasomotion analysis reveals novel arteriole physiological features and progressive modulation of cerebral vascular networks by stroke.

Spontaneous cerebral vasomotion, characterized by ∼0.1 Hz rhythmic contractility, is crucial for brain homeostasis. However, our understanding of vasomotion is limited due to a lack of high-precision analytical methods to determine single vasomotion events at basal levels. Here, we developed a novel strategy that integrates a baseline smoothing algorithm, allowing precise measurements of vasodynamics and concomitant Ca2+ dynamics in mouse cerebral vasculature imaged by two-photon microscopy. We identified several previously unrecognized vasomotion properties under different physiological and pathological conditions, especially in ischemic stroke, which is a highly harmful brain disease that results from vessel occlusion. First, the dynamic characteristics between SMCs Ca2+ and corresponding arteriolar vasomotion are correlated. Second, compared to previous diameter-based estimations, our radius-based measurements reveal anisotropic vascular movements, enabling a more precise determination of the latency between smooth muscle cell (SMC) Ca2+ activity and vasoconstriction. Third, we characterized single vasomotion event kinetics at scales of less than 4 seconds. Finally, following pathological vasoconstrictions induced by ischemic stroke, vasoactive arterioles entered an inert state and persisted despite recanalization. In summary, we developed a highly accurate technique for analyzing spontaneous vasomotion, and our data suggested a potential strategy to reduce stroke damage by promoting vasomotion recovery.

Ca2+ oscillation in vascular smooth muscle cells control myogenic spontaneous vasomotion and counteract post-ischemic no-reflow.

Ischemic stroke produces the highest adult disability. Despite successful recanalization, no-reflow, or the futile restoration of the cerebral perfusion after ischemia, is a major cause of brain lesion expansion. However, the vascular mechanism underlying this hypoperfusion is largely unknown, and no approach is available to actively promote optimal reperfusion to treat no-reflow. Here, by combining two-photon laser scanning microscopy (2PLSM) and a mouse middle cerebral arteriolar occlusion (MCAO) model, we find myogenic vasomotion deficits correlated with post-ischemic cerebral circulation interruptions and no-reflow. Transient occlusion-induced transient loss of mitochondrial membrane potential (ΔΨm) permanently impairs mitochondria-endoplasmic reticulum (ER) contacts and abolish Ca2+ oscillation in smooth muscle cells (SMCs), the driving force of myogenic spontaneous vasomotion. Furthermore, tethering mitochondria and ER by specific overexpression of ME-Linker in SMCs restores cytosolic Ca2+ homeostasis, remotivates myogenic spontaneous vasomotion, achieves optimal reperfusion, and ameliorates neurological injury. Collectively, the maintaining of arteriolar myogenic vasomotion and mitochondria-ER contacts in SMCs, are of critical importance in preventing post-ischemic no-reflow.

Fate mapping of Spp1 expression reveals age-dependent plasticity of disease-associated microglia-like cells after brain injury.

Microglial reactivity to injury and disease is emerging as a heterogeneous, dynamic, and crucial determinant in neurological disorders. However, the plasticity and fate of disease-associated microglia (DAM) remain largely unknown. We established a lineage tracing system, leveraging the expression dynamics of secreted phosphoprotein 1（Spp1） to label and track DAM-like microglia during brain injury and recovery. Fate mapping of Spp1+ microglia during stroke in juvenile mice revealed an irreversible state of DAM-like microglia that were ultimately eliminated from the injured brain. By contrast, DAM-like microglia in the neonatal stroke models exhibited high plasticity, regaining a homeostatic signature and integrating into the microglial network after recovery. Furthermore, neonatal injury had a lasting impact on microglia, rendering them intrinsically sensitized to subsequent immune challenges. Therefore, our findings highlight the plasticity and innate immune memory of neonatal microglia, shedding light on the fate of DAM-like microglia in various neuropathological conditions.

Synaptic-like transmission between neural axons and arteriolar smooth muscle cells drives cerebral neurovascular coupling.

Neurovascular coupling (NVC) is important for brain function and its dysfunction underlies many neuropathologies. Although cell-type specificity has been implicated in NVC, how active neural information is conveyed to the targeted arterioles in the brain remains poorly understood. Here, using two-photon focal optogenetics in the mouse cerebral cortex, we demonstrate that single glutamatergic axons dilate their innervating arterioles via synaptic-like transmission between neural-arteriolar smooth muscle cell junctions (NsMJs). The presynaptic parental-daughter bouton makes dual innervations on postsynaptic dendrites and on arteriolar smooth muscle cells (aSMCs), which express many types of neuromediator receptors, including a low level of glutamate NMDA receptor subunit 1 (Grin1). Disruption of NsMJ transmission by aSMC-specific knockout of GluN1 diminished optogenetic and whisker stimulation-caused functional hyperemia. Notably, the absence of GluN1 subunit in aSMCs reduced brain atrophy following cerebral ischemia by preventing Ca2+ overload in aSMCs during arteriolar constriction caused by the ischemia-induced spreading depolarization. Our findings reveal that NsMJ transmission drives NVC and open up a new avenue for studying stroke.

Modeling Distal Middle Cerebral Artery Occlusion in Neonatal Rodents with Magnetic Nanoparticles or Magnetized Red Blood Cells.

It is challenging to establish animal models to mimic perinatal arterial ischemic stroke. Here, we provided two approaches that precisely occlude rodent pups' distal middle cerebral artery of rodent pups at any postnatal age. One uses magnetic nanoparticles to generate platelet-rich thrombus, and the other utilizes magnetized red blood cells (mRBCs) to generate an erythrocyte-rich embolus. Both approaches result in focal cerebral ischemia followed by controllable reperfusion while requiring no arterial surgery.

Proteins secreted by brain arteriolar smooth muscle cells are instructive for neural development.

Intercellular communication between vascular and nerve cells mediated by diffusible proteins has recently emerged as a critical intrinsic program for neural development. However, whether the vascular smooth muscle cell (VSMC) secretome regulates the connectivity of neural circuits remains unknown. Here, we show that conditioned medium from brain VSMC cultures enhances multiple neuronal functions, such as neuritogenesis, neuronal maturation, and survival, thereby improving circuit connectivity. However, protein denaturation by heating compromised these effects. Combined omics analyses of donor VSMC secretomes and recipient neuron transcriptomes revealed that overlapping pathways of extracellular matrix receptor signaling and adhesion molecule integrin binding mediate VSMC-dependent neuronal development. Furthermore, we found that human arterial VSMCs promote neuronal development in multiple ways, including expanding the time window for nascent neurite initiation, increasing neuronal density, and promoting synchronized firing, whereas human umbilical vein VSMCs lack this capability. These in vitro data indicate that brain arteriolar VSMCs may carry direct instructive information for neural development through intercellular communication in vivo.

Dual-expression system for blue fluorescent protein optimization.

Spectrally diverse fluorescent proteins (FPs) provide straightforward means for multiplexed imaging of biological systems. Among FPs fitting standard color channels, blue FPs (BFPs) are characterized by lower brightness compared to other spectral counterparts. Furthermore, available BFPs were not systematically characterized for imaging in cultured mammalian cells and common model organisms. Here we introduce a pair of new BFPs, named Electra1 and Electra2, developed through hierarchical screening in bacterial and mammalian cells using a novel dual-expression vector. We performed systematic benchmarking of Electras against state-of-art BFPs in cultured mammalian cells and demonstrated their utility as fluorescent tags for structural proteins. The Electras variants were validated for multicolor neuroimaging in Caenorhabditis elegans, zebrafish larvae, and mice in comparison with one of the best in the class BFP mTagBFP2 using one-photon and two-photon microscopy. The developed BFPs are suitable for multicolor imaging of cultured cells and model organisms in vivo. We believe that the described dual-expression vector has a great potential to be adopted by protein engineers for directed molecular evolution of FPs.

Precise control of embolic stroke with magnetized red blood cells in mice.

Precise embolism control in immature brains can facilitate mechanistic studies of brain damage and repair after perinatal arterial ischemic stroke (PAIS), but it remains a technical challenge. Microhemorrhagic transformation is observed in one-third of infant patients who have suffered PAIS, but the underlying mechanism remains elusive. Building on an established approach that uses magnetic nanoparticles to induce PAIS, we develop a more advanced approach that utilizes magnetized erythrocytes to precisely manipulate de novo and in situ embolus formation and reperfusion in perinatal rodent brains. This approach grants spatiotemporal control of embolic stroke without any transarterial delivery of pre-formed emboli. Transmission electron microscopy revealed that erythrocytes rather than nanoparticles are the main material obstructing the vessels. Both approaches can induce microbleeds as an age-dependent complication; this complication can be prevented by microglia and macrophage depletion. Thus, this study provides an animal model mimicking perinatal embolic stroke and implies a potential therapeutic strategy for the treatment of perinatal stroke.

PALP: A rapid imaging technique for stratifying ferroptosis sensitivity in normal and tumor tissues in situ.

Ferroptosis is an emerging cancer suppression strategy. However, how to select cancer patients for treating with ferroptosis inducers remains challenging. Here, we develop photochemical activation of membrane lipid peroxidation (PALP), which uses targeted lasers to induce localized polyunsaturated fatty acyl (PUFA)-lipid peroxidation for reporting ferroptosis sensitivity in cells and tissues. PALP captured by BODIPY-C11 can be suppressed by lipophilic antioxidants and iron chelation, and is dependent on PUFA-lipid levels. Moreover, we develop PALPv2, for studying lipid peroxidation on selected membranes along the z axis in live cells using two-photon microscopes. Using PALPv1, we detect PUFA-lipids in multiple tissues, and validate a PUFA-phospholipid reduction during muscle aging as previously reported. Patterns of PALPv1 signals across multiple cancer cell types in vitro and in vivo are concordant with their ferroptosis susceptibility and PUFA-phospholipid levels. We envision that PALP will enable rapid stratification of ferroptosis sensitivity in cancer patients and facilitate PUFA-lipid research.

A Versatile Tiling Light Sheet Microscope for Imaging of Cleared Tissues.

We present a tiling light sheet microscope compatible with all tissue clearing methods for rapid multicolor 3D imaging of cleared tissues with micron-scale (4 × 4 × 10 µm3) to submicron-scale (0.3 × 0.3 × 1 µm3) spatial resolution. The resolving ability is improved to sub-100 nm (70 × 70 × 200 nm3) via tissue expansion. The microscope uses tiling light sheets to achieve higher spatial resolution and better optical sectioning ability than conventional light sheet microscopes. The illumination light is phase modulated to adjust the position and intensity profile of the light sheet based on the desired spatial resolution and imaging speed and to keep the microscope aligned. The ability of the microscope to align via phase modulation alone also ensures its accuracy for multicolor 3D imaging and makes the microscope reliable and easy to operate. Here we describe the working principle and design of the microscope. We demonstrate its utility by imaging various cleared tissues.

Control of occlusion of middle cerebral artery in perinatal and neonatal mice with magnetic force.

Ischemic perinatal stroke (IPS) is common, resulting in significant mortality and morbidity. In such cases, the incidence of unilateral arterial cerebral infarction is often occluded in the middle cerebral artery (MCA), leading to focal ischemia. In adult rodents, blockage of MCA is the most frequently used strategy for ischemic stroke study. However, modeling MCA occlusion (MCAo) in postnatal day 0-7 (P0-7) mouse pups for IPS study has not been accomplished. Here we occluded the dMCA by inducing the accumulation of magnetic particles (MPs) administered through the superficial temporal vein of mice between P0 and P7, which we called neonatal or perinatal SIMPLE (Stroke Induced with Magnetic Particles). SIMPLE produced either permanent or transient occlusion in the dMCA of perinatal and neonatal mice. Permanent MCA occlusion with SIMPLE resulted in cerebral infarction and neuronal death in the brain. SIMPLE can also be used to reliably produce focal ischemic stroke in neonatal or perinatal mouse brains. As a result, SIMPLE allows the modeling of IPS or focal ischemic stroke for further mechanistic studies in mice, with particular utility for mimicking transient focal ischemia in human pre-term babies, which for the first time here has been accomplished in mice.

Control of cerebral ischemia with magnetic nanoparticles.

The precise manipulation of microcirculation in mice can facilitate mechanistic studies of brain injury and repair after ischemia, but this manipulation remains a technical challenge, particularly in conscious mice. We developed a technology that uses micromagnets to induce aggregation of magnetic nanoparticles to reversibly occlude blood flow in microvessels. This allowed induction of ischemia in a specific cortical region of conscious mice of any postnatal age, including perinatal and neonatal stages, with precise spatiotemporal control but without surgical intervention of the skull or artery. When combined with longitudinal live-imaging approaches, this technology facilitated the discovery of a feature of the ischemic cascade: selective loss of smooth muscle cells in juveniles but not adults shortly after onset of ischemia and during blood reperfusion.

The schizophrenia susceptibility gene dysbindin regulates dendritic spine dynamics.

Dysbindin is a schizophrenia susceptibility gene required for the development of dendritic spines. The expression of dysbindin proteins is decreased in the brains of schizophrenia patients, and neurons in mice carrying a deletion in the dysbindin gene have fewer dendritic spines. Hence, dysbindin might contribute to the spine pathology of schizophrenia, which manifests as a decrease in the number of dendritic spines. The development of dendritic spines is a dynamic process involving formation, retraction, and transformation of dendritic protrusions. It has yet to be determined whether dysbindin regulates the dynamics of dendritic protrusions. Here we address this question using time-lapse imaging in hippocampal neurons. Our results show that dysbindin is required to stabilize dendritic protrusions. In dysbindin-null neurons, dendritic protrusions are hyperactive in formation, retraction, and conversion between different types of protrusions. We further show that CaMKIIα is required for the stabilization of mushroom/thin spines, and that the hyperactivity of dendritic protrusions in dysbindin-null neurons is attributed in part to decreased CaMKIIα activity resulting from increased inhibition of CaMKIIα by Abi1. These findings elucidate the function of dysbindin in the dynamic morphogenesis of dendritic protrusions, and reveal the essential roles of dysbindin and CaMKIIα in the stabilization of dendritic protrusions during neuronal development.

Age-dependent regulation of synaptic connections by dopamine D2 receptors.

Dopamine D2 receptors (D2R) are G protein-coupled receptors that modulate synaptic transmission and are important for various brain functions, including learning and working memory. Abnormal D2R signaling has been implicated in psychiatric disorders such as schizophrenia. Here we report a new function of D2R in dendritic spine morphogenesis. Activation of D2R reduced spine number via GluN2B- and cAMP-dependent mechanisms in mice. Notably, this regulation occurred only during adolescence. During this period, D2R overactivation caused by mutations in the schizophrenia risk gene Dtnbp1 led to spine deficiency, dysconnectivity in the entorhinal-hippocampal circuit and impairment of spatial working memory. Notably, these defects could be ameliorated by D2R blockers administered during adolescence. Our findings suggest an age-dependent function of D2R in spine development, provide evidence that D2R dysfunction during adolescence impairs neuronal circuits and working memory, and indicate that adolescent interventions to prevent aberrant D2R activity protect against cognitive impairment.

The novel caspase-3 substrate Gap43 is involved in AMPA receptor endocytosis and long-term depression.

The cysteine protease caspase-3, best known as an executioner of cell death in apoptosis, also plays a non-apoptotic role in N-methyl-d-aspartate receptor-dependent long-term depression of synaptic transmission (NMDAR-LTD) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor endocytosis in neurons. The mechanism by which caspase-3 regulates LTD and AMPA receptor endocytosis, however, remains unclear. Here, we addressed this question by using an enzymatic N-terminal peptide enrichment method and mass spectrometry to identify caspase-3 substrates in neurons. Of the many candidates revealed by this proteomic study, we have confirmed BASP1, Dbn1, and Gap43 as true caspase-3 substrates. Moreover, in hippocampal neurons, Gap43 mutants deficient in caspase-3 cleavage inhibit AMPA receptor endocytosis and LTD. We further demonstrated that Gap43, a protein well-known for its functions in axons, is also localized at postsynaptic sites. Our study has identified Gap43 as a key caspase-3 substrate involved in LTD and AMPA receptor endocytosis, uncovered a novel postsynaptic function for Gap43 and provided new insights into how long-term synaptic depression is induced.

Caspase-3 activation via mitochondria is required for long-term depression and AMPA receptor internalization.

NMDA receptor-dependent synaptic modifications, such as long-term potentiation (LTP) and long-term depression (LTD), are essential for brain development and function. LTD occurs mainly by the removal of AMPA receptors from the postsynaptic membrane, but the underlying molecular mechanisms remain unclear. Here, we show that activation of caspase-3 via mitochondria is required for LTD and AMPA receptor internalization in hippocampal neurons. LTD and AMPA receptor internalization are blocked by peptide inhibitors of caspase-3 and -9. In hippocampal slices from caspase-3 knockout mice, LTD is abolished whereas LTP remains normal. LTD is also prevented by overexpression of the anti-apoptotic proteins XIAP or Bcl-xL, and by a mutant Akt1 protein that is resistant to caspase-3 proteolysis. NMDA receptor stimulation that induces LTD transiently activates caspase-3 in dendrites, without causing cell death. These data indicate an unexpected causal link between the molecular mechanisms of apoptosis and LTD.

Zn2+ mediates ischemia-induced impairment of the ubiquitin-proteasome system in the rat hippocampus.

Abstract Deposition of ubiquitinated protein aggregates is a hallmark of neurodegeneration in both acute neural injuries, such as stroke, and chronic conditions, such as Parkinson's disease, but the underlying mechanisms are poorly understood. In the present study, we examined the role of Zn2+ in ischemia-induced impairment of the ubiquitin-proteasome system in the CA1 region of rat hippocampus after transient global ischemia. We found that scavenging endogenous Zn2+ reduced ischemia-induced ubiquitin conjugation and free ubiquitin depletion. Furthermore, exposure to zinc chloride increased ubiquitination and inhibited proteasomal enzyme activity in cultured hippocampal neurons in a concentration- and time-dependent manner. Further studies of the underlying mechanisms showed that Zn(2+)-induced ubiquitination required p38 activation. These findings indicate that alterations in Zn2+ homeostasis impair the protein degradation pathway.

Brain-derived neurotrophic factor-tropomyosin-related kinase B signaling contributes to activity-dependent changes in synaptic proteins.

The ability of synapses to undergo changes in structure and function in response to alterations of neuronal activity is an essential property of neural circuits. One way that this is achieved is through global changes in the molecular composition of the synapse; however, it is not clear how these changes are coupled to the dynamics of neuronal activity. Here we found that, in cultured rat cortical neurons, bidirectional changes of neuronal activity led to corresponding alterations in the expression of brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor tropomyosin-related kinase B (TrkB), as well as in the level of synaptic proteins. Exogenous BDNF reversed changes in synaptic proteins induced by chronic activity blockade, while inhibiting Trk kinase activity or depleting endogenous BDNF abolished the concentration changes induced by chronic activity elevation. Both tetrodotoxin and bicuculline had significant, but opposite, effects on synaptic protein ubiquitination in a time-dependent manner. Furthermore, exogenous BDNF was sufficient to increase ubiquitination of synaptic proteins, whereas scavenging endogenous BDNF or inhibiting Trk kinase activity prevented the ubiquitination of synaptic proteins induced by chronic elevation of neuronal activity. Inhibiting the proteasome or blocking protein polyubiquitination mimicked the effect of tetrodotoxin on the levels of synaptic proteins and canceled the effects of BDNF. Our study indicates that BDNF-TrkB signaling acts upstream of the ubiquitin proteasome system, linking neuronal activity to protein turnover at the synapse.