Neuropathologists assess vast brain areas to identify diverse and subtly-differentiated morphologies. Standard semi-quantitative scoring approaches, however, are coarse-grained and lack precise ...neuroanatomic localization. We report a proof-of-concept deep learning pipeline that identifies specific neuropathologies-amyloid plaques and cerebral amyloid angiopathy-in immunohistochemically-stained archival slides. Using automated segmentation of stained objects and a cloud-based interface, we annotate > 70,000 plaque candidates from 43 whole slide images (WSIs) to train and evaluate convolutional neural networks. Networks achieve strong plaque classification on a 10-WSI hold-out set (0.993 and 0.743 areas under the receiver operating characteristic and precision recall curve, respectively). Prediction confidence maps visualize morphology distributions at high resolution. Resulting network-derived amyloid beta (Aβ)-burden scores correlate well with established semi-quantitative scores on a 30-WSI blinded hold-out. Finally, saliency mapping demonstrates that networks learn patterns agreeing with accepted pathologic features. This scalable means to augment a neuropathologist's ability suggests a route to neuropathologic deep phenotyping.
Objective
Hyperamylinemia, a common pancreatic disorder in obese and insulin‐resistant patients, is known to cause amylin oligomerization and cytotoxicity in pancreatic islets, leading to β‐cell mass ...depletion and development of type 2 diabetes. Recent data has revealed that hyperamylinemia also affects the vascular system, heart, and kidneys. We therefore hypothesized that oligomerized amylin might accumulate in the cerebrovascular system and brain parenchyma of diabetic patients.
Methods
Amylin accumulation in the brain of diabetic patients with vascular dementia or Alzheimer disease (AD), nondiabetic patients with AD, and age‐matched healthy controls was assessed by quantitative real time polymerase chain reaction, immunohistochemistry, Western blot, and enzyme‐linked immunosorbent assay.
Results
Amylin oligomers and plaques were identified in the temporal lobe gray matter from diabetic patients, but not controls. In addition, extensive amylin deposition was found in blood vessels and perivascular spaces. Intriguingly, amylin deposition was also detected in blood vessels and brain parenchyma of patients with late onset AD without clinically apparent diabetes. Mixed amylin and amyloid β (Aβ) deposits were occasionally observed. However, amylin accumulation leads to amyloid formation independent of Aβ deposition. Tissues infiltrated by amylin showed increased interstitial space, vacuolation, spongiform change, and capillaries bent at amylin accumulation sites. Unlike the pancreas, there was no evidence of amylin synthesis in the brain.
Interpretation
Metabolic disorders and aging promote accumulation of amylin amyloid in the cerebrovascular system and gray matter, altering microvasculature and tissue structure. Amylin amyloid formation in the wall of cerebral blood vessels may also induce failure of elimination of Aβ from the brain, thus contributing to the etiology of AD. Ann Neurol 2013;74:517–526
MECP2, an X-linked gene encoding the epigenetic factor methyl-CpG-binding protein-2, is mutated in Rett syndrome (RTT) and aberrantly expressed in autism. Most children affected by RTT are ...heterozygous Mecp2(-/+) females whose brain function is impaired postnatally due to MeCP2 deficiency. Recent studies suggest a role of glia in causing neuronal dysfunction via a non-cell-autonomous effect in RTT. Here we report a potent neurotoxic activity in the conditioned medium (CM) obtained from Mecp2-null microglia. Hippocampal neurons treated with CM from Mecp2-null microglia showed an abnormal stunted and beaded dendritic morphology, and signs of microtubule disruption and damage of postsynaptic glutamatergic components within 24 h. We identified that the toxic factor in the CM is glutamate, because (1) Mecp2-null microglia released a fivefold higher level of glutamate, (2) blockage of microglial glutamate synthesis by a glutaminase inhibitor abolished the neurotoxic activity, (3) blockage of microglial glutamate release by gap junction hemichannel blockers abolished the neurotoxic activity, and (4) glutamate receptor antagonists blocked the neurotoxicity of the Mecp2-null microglia CM. We further identified that increased levels of glutaminase and connexin 32 in Mecp2-null microglia are responsible for increased glutamate production and release, respectively. In contrast, the CM from highly pure Mecp2-null astrocyte cultures showed no toxic effect. Our results suggest that microglia may influence the onset and progression of RTT and that microglia glutamate synthesis or release could be a therapeutic target for RTT.
Microglia show a rich repertoire of activation patterns regulated by a complex ensemble of surface ion channels, receptors, and transporters. We and others have investigated whether microglia vary ...their K+ channel expression as a means to achieve functional diversity. However, most of the prior studies were conducted using in vitro models such as BV2 cells, primary microglia, or brain slices in culture, which may not accurately reflect microglia physiology in adult individuals. Here we employed an in vivo mouse model of selective innate immune activation by intracerebroventricular injection of lipopolysaccharides (ICV‐LPS) to determine the role of the voltage‐gated Kv1.3 channel in LPS‐induced M1‐like microglial activation. Using microglia acutely isolated from adult brains, we detected Kv1.3 and Kir2.1 currents, and found that ICV‐LPS increased the current density and RNA expression of Kv1.3 but did not affect those of Kir2.1. Genetic knockout of Kv1.3 abolished LPS‐induced microglial activation exemplified by Iba‐1 immunoreactivity and expression of pro‐inflammatory mediators such as IL‐1β, TNF‐α, IL‐6, and iNOS. Moreover, Kv1.3 knockout mitigated the LPS‐induced impairment of hippocampal long‐term potentiation (hLTP), suggesting that Kv1.3 activity regulates pro‐inflammatory microglial neurotoxicity. Pharmacological intervention using PAP‐1, a small molecule that selectively blocks homotetrameric Kv1.3 channels, achieved anti‐inflammatory and hLTP‐recovery effects similar to Kv1.3 knockout. We conclude that Kv1.3 is required for microglial M1‐like pro‐inflammatory activation in vivo. A significant implication of our in vivo data is that Kv1.3 blockers could be therapeutic candidates for neurological diseases where microglia‐mediated neurotoxicity is implicated in the pathogenesis.
Main Points
This in vivo study of the role of Kv1.3 in microglial activation shows that Kv1.3 is required for microglial M1‐like pro‐inflammatory activation and neurotoxicity.
Selective Kv1.3 blockers could be therapeutic candidates for neurological diseases in which neurotoxic microglial activation is implicated.
OBJECTIVE:We determined whether Gram-negative bacterial molecules are associated with Alzheimer disease (AD) neuropathology given that previous studies demonstrate Gram-negative Escherichia coli ...bacteria can form extracellular amyloid and Gram-negative bacteria have been reported as the predominant bacteria found in normal human brains.
METHODS:Brain samples from gray and white matter were studied from patients with AD (n = 24) and age-matched controls (n = 18). Lipopolysaccharide (LPS) and E coli K99 pili protein were evaluated by Western blots and immunocytochemistry. Human brain samples were assessed for E coli DNA followed by DNA sequencing.
RESULTS:LPS and E coli K99 were detected immunocytochemically in brain parenchyma and vessels in all AD and control brains. K99 levels measured using Western blots were greater in AD compared to control brains (p < 0.01) and K99 was localized to neuron-like cells in AD but not control brains. LPS levels were also greater in AD compared to control brain. LPS colocalized with Aβ1-40/42 in amyloid plaques and with Aβ1-40/42 around vessels in AD brains. DNA sequencing confirmed E coli DNA in human control and AD brains.
CONCLUSIONS:E coli K99 and LPS levels were greater in AD compared to control brains. LPS colocalized with Aβ1-40/42 in amyloid plaques and around vessels in AD brain. The data show that Gram-negative bacterial molecules are associated with AD neuropathology. They are consistent with our LPS-ischemia-hypoxia rat model that produces myelin aggregates that colocalize with Aβ and resemble amyloid-like plaques.
Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) promote differentiation into ...classically activated M1‐like microglia, which produce high levels of pro‐inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL‐4 in contrast induces a phenotype associated with anti‐inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K+ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL‐4) microglia and studying their K+ channel expression by whole‐cell patch‐clamp, quantitative PCR and immunohistochemistry. We identified three major types of K+ channels based on their biophysical and pharmacological fingerprints: a use‐dependent, outwardly rectifying current sensitive to the KV1.3 blockers PAP‐1 and ShK‐186, an inwardly rectifying Ba2+‐sensitive Kir2.1 current, and a Ca2+‐activated, TRAM‐34‐sensitive KCa3.1 current. Both KV1.3 and KCa3.1 blockers inhibited pro‐inflammatory cytokine production and iNOS and COX2 expression demonstrating that KV1.3 and KCa3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN‐γ microglia exhibited high KV1.3 current densities (∼50 pA/pF at 40 mV) and virtually no KCa3.1 and Kir currents, while microglia differentiated with IL‐4 exhibited large Kir2.1 currents (∼ 10 pA/pF at −120 mV). KCa3.1 currents were generally low but moderately increased following stimulation with IFN‐γ or ATP (∼10 pS/pF). This differential K+ channel expression pattern suggests that KV1.3 and KCa3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106–121
Main Points
Microglia subtypes differ significantly in the expression of three potassium channels (Kir2.1, KV1.3 and KCa3.1).
KV1.3 and KCa3.1 blockers could be used to inhibit detrimental neuroinflammatory microglia functions.
The goal of this study was to identify the intrinsic links that explain the effect of a Western diet (WD) on cognitive dysfunction. Specific pathogen‐free, wild‐type mice were fed either a control ...diet (CD) or a high‐fat, high‐sucrose WD after weaning and were euthanized at 10 mo of age to study the pathways that affect cognitive health. The results showed that long‐term WD intake reduced hippocampal synaptic plasticity and the level of brain‐derived neurotrophic factor mRNA in the brain and isolated microglia. A WD also activated ERK1/2 and reduced postsynaptic density‐95 in the brain, suggesting postsynaptic damage. Moreover, WD‐fed mice had increased inflammatory signaling in the brain, ileum, liver, adipose tissue, and spleen, which was accompanied by microglia activation. In the brain, as well as in the digestive tract, a WD reduced signaling regulated by retinoic acid and bile acids (BAs), whose receptors form heterodimers to control metabolism and inflammation. Furthermore, a WD intake caused dysbiosis and dysregulated BA synthesis with reduced endogenous ligands for BA receptors, i.e., farnesoid X receptor and G‐protein‐coupled bile acid receptor in the liver and brain. Together, dysregulated BA synthesis and dysbiosis were accompanied by systemic inflammation, microglial activation, and reduced neuroplasticity induced by WD.—Jena, P. K., Sheng, L., Di Lucente, J., Jin, L.‐W., Maezawa, I., Wan, Y.‐J. Y. Dysregulated bile acid synthesis and dysbiosis are implicated in Western diet‐induced systemic inflammation, microglial activation, and reduced neuroplasticity. FASEB J. 32,2866–2877 (2018). www.fasebj.org
Fucosylation, especially core fucosylation of N‐glycans catalyzed by α1‐6 fucosyltransferase (fucosyltransferase 8 or FUT8), plays an important role in regulating the peripheral immune system and ...inflammation. However, its role in microglial activation is poorly understood. Here we used human induced pluripotent stem cells‐derived microglia (hiMG) as a model to study the role of FUT8‐catalyzed core fucosylation in amyloid‐β oligomer (AβO)‐induced microglial activation, in view of its significant relevance to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AβO and lipopolysaccharides (LPS) with a pattern of pro‐inflammatory activation as well as enhanced core fucosylation and FUT8 expression within 24 h. Furthermore, we found increased FUT8 expression in both human AD brains and microglia isolated from 5xFAD mice, a model of AD‐like cerebral amyloidosis. Inhibition of fucosylation in AβO‐stimulated hiMG reduced the induction of pro‐inflammatory cytokines, suppressed the activation of p38MAPK, and rectified phagocytic deficits. Specific inhibition of FUT8 by siRNA‐mediated knockdown also reduced AβO‐induced pro‐inflammatory cytokines. We further showed that p53 binds to the two consensus binding sites in the Fut8 promoter, and that p53 knockdown abolished FUT8 overexpression in AβO‐activated hiMG. Taken together, our evidence supports that FUT8‐catalyzed core fucosylation is a signaling pathway required for AβO‐induced microglia activation and that FUT8 is a component of the p53 signaling cascade regulating microglial behavior. Because microglia are a key driver of AD pathogenesis, our results suggest that microglial FUT8 could be an anti‐inflammatory therapeutic target for AD.
Main Point
Amyloid‐β oligomer‐induced activation of human microglia, a pathogenic process in Alzheimer's disease, requires FUT8‐catalyzed α1‐6 core fucosylation of N‐glycans. FUT8 is a component of the p53 signaling cascade regulating microglial behavior.
TDP-43 inclusions are found in many Alzheimer's disease (AD) patients presenting faster disease progression and greater brain atrophy. Previously, we showed full-length TDP-43 forms spherical ...oligomers and perturbs amyloid-β (Aβ) fibrillization. To elucidate the role of TDP-43 in AD, here, we examined the effect of TDP-43 in Aβ aggregation and the attributed toxicity in mouse models. We found TDP-43 inhibited Aβ fibrillization at initial and oligomeric stages. Aβ fibrillization was delayed specifically in the presence of N-terminal domain containing TDP-43 variants, while C-terminal TDP-43 was not essential for Aβ interaction. TDP-43 significantly enhanced Aβ's ability to impair long-term potentiation and, upon intrahippocampal injection, caused spatial memory deficit. Following injection to AD transgenic mice, TDP-43 induced inflammation, interacted with Aβ, and exacerbated AD-like pathology. TDP-43 oligomers mostly colocalized with intracellular Aβ in the brain of AD patients. We conclude that TDP-43 inhibits Aβ fibrillization through its interaction with Aβ and exacerbates AD pathology.
Significance: 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is currently used for image-guided glioma resection. Typically, this widefield imaging method highlights the ...bulk of high-grade gliomas, but it underperforms at the infiltrating edge where PpIX fluorescence is not visible to the eyes. Fluorescence lifetime imaging (FLIm) has the potential to detect PpIX fluorescence below the visible detection threshold. Moreover, simultaneous acquisition of time-resolved nicotinamide adenine (phosphate) dinucleotide NAD(P)H fluorescence may provide metabolic information from the tumor environment to further improve overall tumor detection.
Aim: We investigate the ability of pulse sampling, fiber-based FLIm to simultaneously image PpIX and NAD(P)H fluorescence of glioma infiltrative margins in patients.
Approach: A mesoscopic fiber-based point-scanning FLIm device (355 nm pulses) was used to simultaneously resolve the fluorescence decay of PpIX (629/53 nm) and NAD(P)H (470/28 nm). The FLIm device enabled data acquisition at room light and rapid (<33 ms) augmentation of FLIm parameters on the surgical field-of-view. FLIm measurements from superficial tumors and tissue areas around the resection margins were performed on three glioblastoma patients in vivo following inspection of PpIX visible fluorescence with a conventional neurosurgical microscope. Microbiopsies were collected from FLIm imaged areas for histopathological evaluation.
Results: The average lifetime from PpIX and NAD(P)H fluorescence distinguished between tumor and surrounding tissue. FLIm measurements of resection margins presented a range of PpIX and NAD(P)H lifetime values (τPpIX ∼ 3 to 14 ns, τNAD(P)H = 3 to 6 ns) associated with unaffected tissue and areas of low-density tumor infiltration.
Conclusions: Intraoperative FLIm could simultaneously detect the emission of PpIX and NAD(P)H from patients in vivo during craniotomy procedures. This approach doubles as a clinical tool to identify tumor areas while performing tissue resection and as a research tool to study tumor microenvironmental changes in vivo. Intraoperative FLIm of 5-ALA-induced PpIX and tissue autofluorescence makes a promising surgical adjunct to guide tumor resection surgery.
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