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Background and purposeThe entorhinal cortex, a critical gateway between the neocortex and hippocampus, is one of the earliest regions affected by Alzheimer disease-associated neurofibrillary tangle pathology. Although our prior work has automatically delineated an MR imaging-based measure of the entorhinal cortex, whether antemortem entorhinal cortex thickness is associated with postmortem tangle burden within the entorhinal cortex is still unknown. Our objective was to evaluate the relationship between antemortem MRI measures of entorhinal cortex thickness and postmortem neuropathological measures.Materials and methodsWe evaluated 50 participants from the Rush Memory and Aging Project with antemortem structural T1-weighted MR imaging and postmortem neuropathologic assessments. Here, we focused on thickness within the entorhinal cortex as anatomically defined by our previously developed MR imaging parcellation system (Desikan-Killiany Atlas in FreeSurfer). Using linear regression, we evaluated the association between entorhinal cortex thickness and tangles and amyloid-β load within the entorhinal cortex and medial temporal and neocortical regions.ResultsWe found a significant relationship between antemortem entorhinal cortex thickness and entorhinal cortex (P = .006) and medial temporal lobe tangles (P = .002); we found no relationship between entorhinal cortex thickness and entorhinal cortex (P = .09) and medial temporal lobe amyloid-β (P = .09). We also found a significant association between entorhinal cortex thickness and cortical tangles (P = .003) and amyloid-β (P = .01). We found no relationship between parahippocampal gyrus thickness and entorhinal cortex (P = .31) and medial temporal lobe tangles (P = .051).ConclusionsOur findings indicate that entorhinal cortex-associated in vivo cortical thinning may represent a marker of postmortem medial temporal and neocortical Alzheimer disease pathology.

We investigated the genetic overlap between Alzheimer's disease (AD) and Parkinson's disease (PD). Using summary statistics (P-values) from large recent genome-wide association studies (GWAS) (total n=89 904 individuals), we sought to identify single nucleotide polymorphisms (SNPs) associating with both AD and PD. We found and replicated association of both AD and PD with the A allele of rs393152 within the extended MAPT region on chromosome 17 (meta analysis P-value across five independent AD cohorts=1.65 × 10(-7)). In independent datasets, we found a dose-dependent effect of the A allele of rs393152 on intra-cerebral MAPT transcript levels and volume loss within the entorhinal cortex and hippocampus. Our findings identify the tau-associated MAPT locus as a site of genetic overlap between AD and PD, and extending prior work, we show that the MAPT region increases risk of Alzheimer's neurodegeneration.

Background: Epidemiological and molecular findings suggest a relationship between Alzheimer's disease (AD) and dyslipidemia, although the nature of this association is not well understood. Results: Using linear mixed effects models, we investigated the relationship between CSF levels of heart fatty acid binding protein (HFABP), a lipid binding protein involved with fatty acid metabolism and lipid transport, amyloid-β (Aβ), phospho-tau, and longitudinal MRI-based measures of brain atrophy among 295 non-demented and demented older individuals. Across all participants, we found a significant association of CSF HFABP with longitudinal atrophy of the entorhinal cortex and other AD-vulnerable neuroanatomic regions. However, we found that the relationship between CSF HABP and brain atrophy was significant only among those with low CSF Aβ1-42and occurred irrespective of phospho-tau181pstatus. Conclusions: Our findings indicate that Aβ-associated volume loss occurs in the presence of elevated HFABP irrespective of phospho-tau. This implicates a potentially important role for fatty acid binding proteins in Alzheimer's disease neurodegeneration. © 2013 Desikan et al.; licensee BioMed Central Ltd.

The apolipoprotein E ε4 gene is the most important genetic risk factor for sporadic Alzheimer's disease, but the link between this gene and neurodegeneration remains unclear. Using array tomography, we analysed >50000 synapses in brains of 11 patients with Alzheimer's disease and five non-demented control subjects and found that synapse loss around senile plaques in Alzheimer's disease correlates with the burden of oligomeric amyloid-β in the neuropil and that this synaptotoxic oligomerized peptide is present at a subset of synapses. Further analysis reveals apolipoprotein E ε4 patients with Alzheimer's disease have significantly higher oligomeric amyloid-β burden and exacerbated synapse loss around plaques compared with apolipoprotein E ε3 patients. Apolipoprotein E4 protein colocalizes with oligomeric amyloid-β and enhances synaptic localization of oligomeric amyloid-β by >5-fold. Biochemical characterization shows that the amyloid-β enriched at synapses by apolipoprotein E4 includes sodium dodecyl sulphate-stable dimers and trimers. In mouse primary neuronal culture, lipidated apolipoprotein E4 enhances oligomeric amyloid-β association with synapses via a mechanism involving apolipoprotein E receptors. Together, these data suggest that apolipoprotein E4 is a co-factor that enhances the toxicity of oligomeric amyloid-β both by increasing its levels and directing it to synapses, providing a link between apolipoprotein E ε4 genotype and synapse loss, a major correlate of cognitive decline in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Anti-amyloid-beta immunization leads to amyloid clearance in patients with Alzheimer's disease, but the effect of vaccination on amyloid-beta-induced neuronal pathology has not been quantitatively examined. The objectives of this study were to address the effects of anti-amyloid-beta active immunization on neurite trajectories and the pathological hallmarks of Alzheimer's disease in the human hippocampus. Hippocampal sections from five patients with Alzheimer's disease enrolled in the AN1792 Phase 2a trial were compared with those from 13 non-immunized Braak-stage and age-matched patients with Alzheimer's disease, and eight age-matched non-demented controls. Analyses included neurite curvature ratio as a quantitative measure of neuritic abnormalities, amyloid and tau loads, and a quantitative characterization of plaque-associated neuritic dystrophy and astrocytosis. Amyloid load and density of dense-core plaques were decreased in the immunized group compared to non-immunized patients (P < 0.01 and P < 0.001, respectively). The curvature ratio in non-immunized patients with Alzheimer's disease was elevated compared to non-demented controls (P < 0.0001). In immunized patients, however, the curvature ratio was normalized when compared to non-immunized patients (P < 0.0001), and not different from non-demented controls. In the non-immunized patients, neurites close to dense-core plaques (within 50 microm) were more abnormal than those far from plaques (i.e. beyond 50 microm) (P < 0.0001). By contrast, in the immunized group neurites close to and far from the remaining dense-core plaques did not differ, and both were straighter compared to the non-immunized patients (P < 0.0001). Compared to non-immunized patients, dense-core plaques remaining after immunization had similar degree of astrocytosis (P = 0.6060), more embedded dystrophic neurites (P < 0.0001) and were more likely to have mitochondrial accumulation (P < 0.001). In addition, there was a significant decrease in the density of paired helical filament-1-positive neurons in the immunized group as compared to the non-immunized (P < 0.05), but not in the density of Alz50 or thioflavin-S positive tangles, suggesting a modest effect of anti-amyloid-beta immunization on tangle pathology. Clearance of amyloid plaques upon immunization with AN1792 effectively improves a morphological measure of neurite abnormality in the hippocampus. This improvement is not just attributable to the decrease in plaque load, but also occurs within the halo of the remaining dense-core plaques. However, these remaining plaques still retain some of their toxic potential. Anti-amyloid-beta immunization might also ameliorate the hippocampal tau pathology through a decrease in tau phosphorylation. These data agree with preclinical animal studies and further demonstrate that human anti-amyloid-beta immunization does not merely clear amyloid from the Alzheimer's disease brain, but reduces some of the neuronal alterations that characterize Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Focuses on the loss of cortical neurons due to normal aging in humans and in non-human primates. Information on the underpinning of age-related deficits; Examinations on the effects of aging; Determination of the neuronal densities.

Diet is a potentially modifiable neurodegenerative disease risk factor. We studied the effects of a typical Western diet (WD; high in refined carbohydrates, cholesterol and saturated fat), relative to a heart-healthy diet (HHD; high in unrefined carbohydrates, polyunsaturated fat and fiber, and low in cholesterol) on brain microvessel transcriptomics and brain metabolomics of the temporal region in Ossabaw minipigs. Thirty-two pigs (16 male and 16 females) were fed a WD or HHD starting at the age of 4 months for a period of 6 months. The WD and HHD were isocaloric and had a similar macronutrient content but differed in macronutrient quality. Within each dietary group, half of the pigs also received atorvastatin. Relative to HHD-fed pigs, WD-fed pigs had 175 genes differentially expressed (fold change > 1.3, FDR < 0.05) by diet, 46 upregulated and 129 downregulated. Gene Set Enrichment Analysis identified 22 gene sets enriched in WD-fed pigs, comprising pathways related to inflammation, angiogenesis, and apoptosis, and 53 gene sets enriched in the HHD-fed pigs, including cell energetics, neurotransmission, and inflammation resolution pathways. Metabolite analysis showed enrichment in arginine, tyrosine, and lysine in WD-fed pigs, and ergothioneine and S-adenosyl methionine in HHD-fed pigs. Atorvastatin treatment did not affect gene expression. These results suggest a likely contribution of diet to brain pathologies characterized by neuroinflammation and neurodegeneration., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics declarations: Animal study was approved by the Tufts Medical Center/Tufts University Institutional Animal Care and Use Committee and all experiments were performed in accordance with the committee guidelines and regulations. Authors have complied with the ARRIVE guidelines., (© 2024. The Author(s).)

Tau protein plays a central role in many neuropathies. The trajectory by which tau spreads through neural networks is disease-specific but the events driving progression are unknown. This is due in part to the challenge of characterizing tau aggregates in situ . We address that challenge using in situ micro-x-ray diffraction (µXRD) and micro-X-ray fluorescence (μXRF) to examine tau lesions in the brain of a 79-year-old male with dementia. Neuropathological examination revealed classical forms of tau in the hippocampal formation: extensive Pick bodies in the granular layer; modest numbers of neurofibrillary tangles and dystrophic neurites in the CA4 and hilus. µXRD indicated that Pick bodies are low in fibril content, whereas neurofibrillary lesions within adjacent tissue exhibit far greater density of fibrillar tau. μXRF demonstrated elevated levels of zinc, calcium and phosphorous in all tau-containing lesions whereas sulfur deposition was greatest in lesions exhibiting high fibrillar content. Correlation of lesion morphology with anatomical localization, tau fibrillation and differential elemental accumulation suggests tau fibrils generate biochemically distinct microenvironments that influence lesion morphology, tau seed formation and spreading., Competing Interests: Competing Interest Statement: There are no competing interests.

Intracellular neurofibrillary tangles that consist of misfolded tau protein 1 cause neurodegeneration in Alzheimer's disease (AD) and frontotemporal dementia (FTD). Tau pathology spreads cell-to-cell 2 but the exact mechanisms of tau release and intercellular transmission remain poorly defined. Tau is released from neurons as free protein or in extracellular vesicles (EVs) 3-5 but the role of these different release mechanisms in intercellular tau transmission is unclear. Here, we show that the neuronal gene Arc is critical for packaging tau into EVs. Brain EVs purified from human tau (hTau) transgenic rTg4510 mice (rTg WT ) contain high levels of hTau that are capable of seeding tau pathology. In contrast, EVs purified from rTg WT crossed with Arc knock-out mice (rTg Arc KO ) have significantly less hTau and cannot seed tau aggregation. Arc facilitates the release of hTau in EVs produced via the I-BAR protein IRSp53, but not free tau. Arc protein directly binds hTau to form a fuzzy complex that we identified in both mouse and human brain tissue. We find that pathological intracellular hTau accumulates in neurons in rTg Arc KO mice, which correlates with accelerated neuron loss in the hippocampus. Finally, we find that intercellular tau transmission is significantly abrogated in Arc KO mice. We conclude that Arc-dependent release of tau in EVs plays a significant role in intracellular tau elimination and intercellular tau transmission.

Biological and clinical heterogeneity is a major challenge in research for developing new treatments for Alzheimer's disease (AD). AD may be defined by its amyloid beta and tau pathologies, but we recognize that mixed pathologies are common, and that diverse genetics, central nervous system (CNS) and systemic pathophysiological processes, and environmental/experiential factors contribute to AD's diverse clinical and neuropathological features. All these factors are rational targets for therapeutic development; indeed, there are hundreds of candidate pharmacological, dietary, neurostimulation, and lifestyle interventions that show benefits in homogeneous laboratory models. Conventional clinical trial designs accommodate heterogeneity poorly, and this may be one reason that progress in translating candidate interventions has been so difficult. We review the challenges of AD's heterogeneity for the clinical trials enterprise. We then discuss how advances in repeatable biomarkers and digital phenotyping enable novel "single-case" and adaptive trial designs to accelerate therapeutics development, moving us closer to personalized research and medicine for AD. HIGHLIGHTS: Alzheimer's disease is diverse in its clinical features, course, risks, and biology. Typical randomized controlled trials are exclusive and necessarily large to attain arm comparability with broad outcomes. Repeated blood biomarkers and digital tracking can improve outcome measure precision and sensitivity. This enables the use of novel "single-case" and adaptive trial designs for inclusivity, rigor, and efficiency., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Pathological tau fibrils in progressive supranuclear palsy, frontotemporal dementia, chronic traumatic encephalopathy, and Alzheimer's disease each have unique conformations, and post-translational modifications that correlate with unique disease characteristics. However, within Alzheimer's disease (AD), both fibrillar (sarkosyl insoluble (AD SARK tau)), and nonfibrillar (aqueous extractable high molecular weight (AD HMW tau)) preparations have been suggested to be seed-competent. We now explore if these preparations are similar or distinct in their in vivo seeding characteristics. Using an in vivo amplification and time-course paradigm we demonstrate that, for AD HMW and AD SARK tau species, the amplified material is biochemically similar to the original sample. The HMW and SARK materials also show different clearance, propagation kinetics, and propagation patterns. These data indicate the surprising co-occurrence of multiple distinct tau species within the same AD brain, supporting the idea that multiple tau conformers - both fibrillar and nonfibrillar- can impact phenotype in AD., (© 2024. The Author(s).)

A prevailing hypothesis is that neurofibrillary tangles play a causal role in driving cognitive decline in Alzheimer's disease (AD) because tangles correlate anatomically with areas that undergo neuronal loss. We used two-photon longitudinal imaging to directly test this hypothesis and observed the fate of individual neurons in two mouse models. At any time point, neurons without tangles died at >3 times the rate as neurons with tangles. Additionally, prior to dying, they became >20% more distant from neighboring neurons across imaging sessions. Similar microstructural changes were evident in a population of non-tangle-bearing neurons in Alzheimer's donor tissues. Together, these data suggest that nonfibrillar tau puts neurons at high risk of death, and surprisingly, the presence of a tangle reduces this risk. Moreover, cortical microstructure changes appear to be a better predictor of imminent cell death than tangle status is and a promising tool for identifying dying neurons in Alzheimer's., Competing Interests: Declaration of interests B.T.H. has a family member who works at Novartis and owns stock in Novartis; he serves on the SAB of Dewpoint and owns stock. He serves on a scientific advisory board or is a consultant for AbbVie, Avrobio, Axon, Biogen, BMS Cell Signaling, Genentech, Ionis, Novartis, Seer, Takeda, the United States Department of Justice, and Vigil, Voyager. His laboratory is supported by sponsored research agreements with AbbVie and F Prime and research grants from the Cure Alzheimer’s Fund, Tau Consortium, and the JPB Foundation. R.E.B. works on the AbbVie-Hyman Collaboration., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Progressive supranuclear palsy (PSP) is a neurodegenerative disease characterized by 4R tau deposition in neurons as well as in astrocytes and oligodendrocytes. While astrocytic tau deposits are rarely observed in normal aging (so-called aging-related tau astrogliopathy, ARTAG) and Alzheimer's disease (AD), astrocytic tau in the form of tufted astrocytes is a pathognomonic hallmark of PSP. Classical biochemical experiments emphasized tau synthesis in neurons in the central nervous system, suggesting that astrocytic tau inclusions might be derived from uptake of extracellular neuronal-derived tau. However, recent single-nucleus RNAseq experiments highlight the fact that MAPT, the gene encoding tau, is also expressed by astrocytes, albeit in lower amounts. We, therefore, revisited the question of whether astrocyte-driven expression of tau might contribute to astrocytic tau aggregates in PSP by performing fluorescent in situ hybridization/immunohistochemical co-localization in human postmortem brain specimens from individuals with PSP and AD with ARTAG as well as normal controls. We find that, in PSP but not in AD, tau-immunoreactive astrocytes have higher levels of MAPT mRNA compared to astrocytes that do not have tau aggregates. These results suggest that astrocytic responses in PSP are unique to this tauopathy and support the possibility that fundamental changes in PSP astrocyte-endogenous mRNA biology contribute to increased synthesis of tau protein and underlies the formation of the astrocytic tau deposits characteristic of PSP., (© 2024. The Author(s).)

For the past three decades, apolipoprotein E (APOE) has been known as the single greatest genetic modulator of sporadic Alzheimer disease (AD) risk, influencing both the average age of onset and the lifetime risk of developing AD. The APOEε4 allele significantly increases AD risk, whereas the ε2 allele is protective relative to the most common ε3 allele. However, large differences in effect size exist across ethnoracial groups that are likely to depend on both global genetic ancestry and local genetic ancestry, as well as gene-environment interactions. Although early studies linked APOE to amyloid-β - one of the two culprit aggregation-prone proteins that define AD - in the past decade, mounting work has associated APOE with other neurodegenerative proteinopathies and broader ageing-related brain changes, such as neuroinflammation, energy metabolism failure, loss of myelin integrity and increased blood-brain barrier permeability, with potential implications for longevity and resilience to pathological protein aggregates. Novel mouse models and other technological advances have also enabled a number of therapeutic approaches aimed at either attenuating the APOEε4-linked increased AD risk or enhancing the APOEε2-linked AD protection. This Review summarizes this progress and highlights areas for future research towards the development of APOE-directed therapeutics., (© 2024. Springer Nature Limited.)

In Alzheimer's disease (AD), the microtubule-binding protein tau becomes abnormally hyperphosphorylated and aggregated in selective brain regions such as the cortex and hippocampus 1-3 . However, other brain regions like the cerebellum and brain stem remain largely intact despite the universal expression of tau throughout the brain. Here, we found that an understudied splice isoform of tau termed "big tau" is significantly more abundant in the brain regions less vulnerable to tau pathology compared to tau pathology-vulnerable regions. We used various cellular and animal models to demonstrate that big tau possesses multiple properties that can resist AD-related pathological changes. Importantly, human AD patients show a higher expression level of pathology-resisting big tau in the cerebellum, the brain region spared from tau pathology. Our study examines the unique properties of big tau, expanding our current understanding of tau pathophysiology. Altogether, our data suggest that alternative splicing to favor big tau is a viable strategy to modulate tau pathology.

Deciphering the striatal interneuron diversity is key to understanding the basal ganglia circuit and to untangling the complex neurological and psychiatric diseases affecting this brain structure. We performed snRNA-seq and spatial transcriptomics of postmortem human caudate nucleus and putamen samples to elucidate the diversity and abundance of interneuron populations and their inherent transcriptional structure in the human dorsal striatum. We propose a comprehensive taxonomy of striatal interneurons with eight main classes and fourteen subclasses, providing their full transcriptomic identity and spatial expression profile as well as additional quantitative FISH validation for specific populations. We have also delineated the correspondence of our taxonomy with previous standardized classifications and shown the main transcriptomic and class abundance differences between caudate nucleus and putamen. Notably, based on key functional genes such as ion channels and synaptic receptors, we found matching known mouse interneuron populations for the most abundant populations, the recently described PTHLH and TAC3 interneurons. Finally, we were able to integrate other published datasets with ours, supporting the generalizability of this harmonized taxonomy., (© 2024. The Author(s).)

We present open-source tools for three-dimensional (3D) analysis of photographs of dissected slices of human brains, which are routinely acquired in brain banks but seldom used for quantitative analysis. Our tools can: (1) 3D reconstruct a volume from the photographs and, optionally, a surface scan; and (2) produce a high-resolution 3D segmentation into 11 brain regions per hemisphere (22 in total), independently of the slice thickness. Our tools can be used as a substitute for ex vivo magnetic resonance imaging (MRI), which requires access to an MRI scanner, ex vivo scanning expertise, and considerable financial resources. We tested our tools on synthetic and real data from two NIH Alzheimer's Disease Research Centers. The results show that our methodology yields accurate 3D reconstructions, segmentations, and volumetric measurements that are highly correlated to those from MRI. Our method also detects expected differences between post mortem confirmed Alzheimer's disease cases and controls. The tools are available in our widespread neuroimaging suite 'FreeSurfer' (https://surfer.nmr.mgh.harvard.edu/fswiki/PhotoTools)., Competing Interests: HG, HT, BB, YB, JW, RH, LD, AC, EM, CL, MK, MM, ER, EB, MM, TC, DO, MF, SY, KV, AD, CM, CK, JI No competing interests declared, BF BF has a financial interest in CorticoMetrics, a company developing brain MRI measurementtechnology; his interests are reviewed and managed by Massachusetts General Hospital, BH Reviewing editor, eLife, (© 2023, Gazula et al.)

Sortilin-related receptor 1 (SORL1) is an intracellular sorting receptor genetically implicated in Alzheimer's disease (AD) that impacts amyloid precursor protein trafficking. The objective of these studies was to test the hypothesis that SORL1 binds tau, modulates its cellular trafficking and impacts the aggregation of cytoplasmic tau induced by pathological forms of tau. Using surface plasmon resonance measurements, we observed high-affinity binding of tau to SORL1 and the vacuolar protein sorting 10 domain of SORL1. Interestingly, unlike LDL receptor-related protein 1, SORL1 binds tau at both pH 7.4 and pH 5.5, revealing its ability to bind tau at endosomal pH. Immunofluorescence studies confirmed that exogenously added tau colocalized with SORL1 in H4 neuroglioma cells, while overexpression of SORL1 in LDL receptor-related protein 1-deficient Chinese hamster ovary (CHO) cells resulted in a marked increase in the internalization of tau, indicating that SORL1 can bind and mediate the internalization of monomeric forms of tau. We further demonstrated that SORL1 mediates tau seeding when tau RD P301S FRET biosensor cells expressing SORL1 were incubated with high molecular weight forms of tau isolated from the brains of patients with AD. Seeding in H4 neuroglioma cells is significantly reduced when SORL1 is knocked down with siRNA. Finally, we demonstrate that the N1358S mutant of SORL1 significantly increases tau seeding when compared to WT SORL1, identifying for the first time a potential mechanism that connects this specific SORL1 mutation to Alzheimer's disease. Together, these studies identify SORL1 as a receptor that contributes to trafficking and seeding of pathogenic tau., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite its high prevalence among dementias, Lewy body dementia (LBD) remains poorly understood with a limited, albeit growing, evidence base. The public-health burden that LBD imposes is worsened by overlapping pathologies, which contribute to misdiagnosis, and lack of treatments. For this report, we gathered and analyzed public-domain information on advocacy, funding, research outputs, and the therapeutic pipeline to identify gaps in each of these key elements. To further understand the current gaps, we also conducted interviews with leading experts in regulatory/governmental agencies, LBD advocacy, academic research, and biopharmaceutical research, as well as with funding sources. We identified wide gaps across the entire landscape, the most critical being in research. Many of the experts participated in a workshop to discuss the prioritization of research areas with a view to accelerating therapeutic development and improving patient care. This white paper outlines the opportunities for bridging the major LBD gaps and creates the framework for collaboration in that endeavor. HIGHLIGHTS: A group representing academia, government, industry, and consulting expertise was convened to discuss current progress in Dementia with Lewy Body care and research. Consideration of expert opinion,natural language processing of the literature as well as publicly available data bases, and Delphi inspired discussion led to a proposed consensus document of priorities for the field., (© 2024 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Introduction: Omics studies have revealed that various brain cell types undergo profound molecular changes in Alzheimer's disease (AD) but the spatial relationships with plaques and tangles and APOE-linked differences remain unclear., Methods: We performed laser capture microdissection of amyloid beta (Aβ) plaques, the 50 μm halo around them, tangles with the 50 μm halo around them, and areas distant (> 50 μm) from plaques and tangles in the temporal cortex of AD and control donors, followed by RNA-sequencing., Results: Aβ plaques exhibited upregulated microglial (neuroinflammation/phagocytosis) and downregulated neuronal (neurotransmission/energy metabolism) genes, whereas tangles had mostly downregulated neuronal genes. Aβ plaques had more differentially expressed genes than tangles. We identified a gradient Aβ plaque > peri-plaque > tangle > distant for these changes. AD APOE ε4 homozygotes had greater changes than APOE ε3 across locations, especially within Aβ plaques., Discussion: Transcriptomic changes in AD consist primarily of neuroinflammation and neuronal dysfunction, are spatially associated mainly with Aβ plaques, and are exacerbated by the APOE ε4 allele., (© 2023 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Idiopathic normal pressure hydrocephalus is a disorder of unknown pathophysiology whose diagnosis is paradoxically made by a positive response to its proposed treatment with cerebrospinal fluid diversion. There are currently no idiopathic normal pressure hydrocephalus disease genes or biomarkers. A systematic analysis of familial idiopathic normal pressure hydrocephalus could aid in clinical diagnosis, prognosis, and treatment stratification, and elucidate disease patho-etiology. In this 2-part analysis, we review literature-based evidence for inheritance of idiopathic normal pressure hydrocephalus in 22 pedigrees, and then present a novel case series of 8 familial idiopathic normal pressure hydrocephalus patients. For the case series, demographics, familial history, pre- and post-operative symptoms, and cortical pathology were collected. All novel familial idiopathic normal pressure hydrocephalus patients exhibited improvement following shunt treatment and absence of neurodegenerative cortical pathology (amyloid-beta and hyperphosphorylated tau), in contrast to many sporadic cases of idiopathic normal pressure hydrocephalus with variable clinical responses. Analysis of the 30 total familial idiopathic normal pressure hydrocephalus cases reported herein is highly suggestive of an autosomal dominant mechanism of inheritance. This largest-ever presentation of multiply affected idiopathic normal pressure hydrocephalus pedigrees provides strong evidence for Mendelian inheritance and autosomal dominant transmission of an idiopathic normal pressure hydrocephalus trait in a subset of patients that positively respond to shunting and lack neurodegenerative pathology. Genomic investigation of these families may identify the first bona fide idiopathic normal pressure hydrocephalus disease gene., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A key hallmark of Alzheimer's disease (AD) is the accumulation of hyperphosphorylated tau in neurofibrillary tangles. This occurs alongside neuroinflammation and neurodegeneration. Pathological tau propagates through the AD brain in a defined manner, which correlates with neuron and synapse loss and cognitive decline. One proposed mechanism of tau spread is through synaptically connected brain structures. Apolipoprotein E4 ( APOE4 ) genotype is the strongest genetic risk factor for late-onset AD and is associated with increased tau burden. Whether the apolipoprotein E ( APOE ) genotype influences neurodegeneration via tau spread is currently unknown. Here, we demonstrate that virally expressed human tau (with the P301L mutation) injected into mouse entorhinal cortex at 5-6 months or 15-16 months of age spreads trans-synaptically to the hippocampus by 14 weeks post-injection. Injections of tau in mice expressing human APOE2 , APOE3 or APOE4 , as well as APOE knock-outs, showed that tau can spread trans-synaptically in all genotypes and that APOE genotype and age do not affect the spread of tau. These data suggest that APOE genotype is not directly linked to synaptic spread of tau in our model, but other mechanisms involving non-cell autonomous manners of tau spread are still possible., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship and/or publication of this article: T.L.S.-J. receives funding as a scientific advisory board member/scientific consultant from Cognition Therapeutics, Scottish Brain Sciences, and Jay Therapeutics., (© The Author(s) 2023.)

Background: The prion-like propagation of tau in neurodegenerative disorders implies that misfolded pathological tau can recruit the normal protein and template its aggregation. Here, we report the methods for the development of sensitive biosensor cell lines for the detection of tau seeding activity., Results: We performed the rational design of novel tau probes based on the current structural knowledge of pathological tau aggregates in Alzheimer's disease. We generated Förster resonance energy transfer (FRET)-based biosensor stable cell lines and characterized their sensitivity, specificity, and overall ability to detect bioactive tau in human samples. As compared to the reference biosensor line, the optimized probe design resulted in an increased efficiency in the detection of tau seeding. The increased sensitivity allowed for the detection of lower amount of tau seeding competency in human brain samples, while preserving specificity for tau seeds found in Alzheimer's disease., Conclusions: This next generation of FRET-based biosensor cells is a novel tool to study tau seeding activity in Alzheimer's disease human samples, especially in samples with low levels of seeding activity, which may help studying early tau-related pathological events., (© 2023. Editorial Group and BioMed Central Ltd., part of Springer Nature.)

Recombinant adeno-associated viral vectors (rAAVs) are a promising strategy to treat neurodegenerative diseases because of their ability to infect non-dividing cells and confer long-term transgene expression. Despite an ever-growing library of capsid variants, widespread delivery of AAVs in the adult central nervous system remains a challenge. We have previously demonstrated successful distribution of secreted proteins by infection of the ependyma, a layer of post-mitotic epithelial cells lining the ventricles of the brain and central column of the spinal cord, and subsequent protein delivery via the cerebrospinal fluid (CSF). Here we define a functional ependyma promoter to enhance expression from this cell type. Using RNA sequencing on human autopsy samples, we identified disease- and age-independent ependyma gene signatures. Associated promoters were cloned and screened as libraries in mouse and rhesus macaque to reveal cross-species function of a human DNA-derived von Willebrand factor domain containing 3A ( VWA3A ) promoter. When tested in mice, our VWA3A promoter drove strong, ependyma-localized expression of eGFP and increased secreted ApoE protein levels in the CSF by 2-12× over the ubiquitous iCAG promoter., Competing Interests: B.L.D. serves an advisory role with equity in Latus Biosciences, Patch Bio, Voyager Therapeutics, Carbon Biosciences, Spirovant Biosciences, Resilience, Panorama Medicines, Saliogen, and Homology Medicines. She has sponsored research from Novartis, Roche, Latus, Homology Medicines, Saliogen, and Spirovant. B.T.H. is on the scientific advisory board of Latus Bio and has an equity interest. He has a family member who works at Novartis and owns stock in Novartis; he serves on the SAB of Dewpoint and owns stock. He serves on a scientific advisory board or is a consultant for AbbVie, Aprinoia Therapeutics, Arvinas, Avrobio, Axial, Biogen, BMS, Cure Alz Fund, Cell Signaling, Dewpoint, Eisai, Genentech, Ionis, Latus, Novartis, Sangamo, Sanofi, Seer, Takeda, the US Department of Justice, Vigil, and Voyager., (© 2023 The Authors.)

Vascular endothelial cells play an important role in maintaining brain health, but their contribution to Alzheimer's disease (AD) is obscured by limited understanding of the cellular heterogeneity in normal aged brain and in disease. To address this, we performed single nucleus RNAseq on tissue from 32 human AD and non-AD donors (19 female, 13 male) each with five cortical regions: entorhinal cortex, inferior temporal gyrus, prefrontal cortex, visual association cortex, and primary visual cortex. Analysis of 51,586 endothelial cells revealed unique gene expression patterns across the five regions in non-AD donors. Alzheimer's brain endothelial cells were characterized by upregulated protein folding genes and distinct transcriptomic differences in response to amyloid β plaques and cerebral amyloid angiopathy. This dataset demonstrates previously unrecognized regional heterogeneity in the endothelial cell transcriptome in both aged non-AD and AD brain. SIGNIFICANCE STATEMENT In this work, we show that vascular endothelial cells collected from five different brain regions display surprising variability in gene expression. In the presence of Alzheimer's disease pathology, endothelial cell gene expression is dramatically altered with clear differences in regional and temporal changes. These findings help explain why certain brain regions appear to differ in susceptibility to disease-related vascular remodeling events that may impact blood flow., (Copyright © 2023 Bryant et al.)

Unique strains of α-synuclein aggregates have been postulated to underlie the spectrum of clinical and pathological presentations seen across the synucleinopathies. Whereas multiple system atrophy (MSA) is associated with a predominance of oligodendroglial α-synuclein inclusions, α-synuclein aggregates in Parkinson's disease (PD) preferentially accumulate in neurons. The G51D mutation in the SNCA gene encoding α-synuclein causes an aggressive, early-onset form of PD that exhibits clinical and neuropathological traits reminiscent of both PD and MSA. To assess the strain characteristics of G51D PD α-synuclein aggregates, we performed propagation studies in M83 transgenic mice by intracerebrally inoculating patient brain extracts. The properties of the induced α-synuclein aggregates in the brains of injected mice were examined using immunohistochemistry, a conformational stability assay, and by performing α-synuclein seed amplification assays. Unlike MSA-injected mice, which developed a progressive motor phenotype, G51D PD-inoculated animals remained free of overt neurological illness for up to 18 months post-inoculation. However, a subclinical synucleinopathy was present in G51D PD-inoculated mice, characterized by the accumulation of α-synuclein aggregates in restricted regions of the brain. The induced α-synuclein aggregates in G51D PD-injected mice exhibited distinct properties in a seed amplification assay and were much more stable than those present in mice injected with MSA extract, which mirrored the differences observed between human MSA and G51D PD brain samples. These results suggest that the G51D SNCA mutation specifies the formation of a slowly propagating α-synuclein strain that more closely resembles α-synuclein aggregates associated with PD than MSA., (© 2023. The Author(s).)

Background: Visium Spatial Gene Expression (ST) is a method combining histological spatial information with transcriptomics profiles directly from tissue sections. The use of spatial information has made it possible to discover new modes of gene expression regulations. However, in the ST experiment, the nucleus size of cells may exceed the thickness of a tissue slice. This may, in turn, negatively affect comprehensive capturing the transcriptomics profile in a single slice, especially for tissues having large differences in the size of nuclei., Methods: Here, we defined the effect of Consecutive Slices Data Integration (CSDI) on unveiling accurate spot clustering and deconvolution of spatial transcriptomic spots in human postmortem brains. By considering the histological information as reference, we assessed the improvement of unsupervised clustering and single nuclei RNA-seq and ST data integration before and after CSDI., Results: Apart from the escalated number of defined clusters representing neuronal layers, the pattern of clusters in consecutive sections was concordant only after CSDI. Besides, the assigned cell labels to spots matches the histological pattern of tissue sections after CSDI., Conclusion: CSDI can be applied to investigate consecutive sections studied with ST in the human cerebral cortex, avoiding misinterpretation of spot clustering and annotation, increasing accuracy of cell recognition as well as improvement in uncovering the layers of grey matter in the human brain., (© 2023. The Author(s).)

Studies of post-mortem human tissue provide insight into pathological processes, but are inherently limited by practical considerations that limit the scale at which tissue can be examined, and the obvious issue that the tissue reflects only one time point in a continuous disease process. We approached this problem by adapting new tissue clearance techniques to an entire cortical area of human brain, which allows surveillance of hundreds of thousands of neurons throughout the depth of the entire cortical thickness. This approach allows detection of 'rare' events that may be difficult to detect in standard 5 micrometre-thick paraffin sections. For example, it is well established that neurofibrillary tangles begin within a neuron, and ultimately, in at least some instances, persist in the brain even after the neuron has died. These are referred to as 'ghost tangles', a term that appropriately implies their 'difficult to see' ephemeral qualities. We set out to find ghost tangles as one example of the power of the tissue clearance/image analysis techniques to detect rare events, and to learn what happens at the end-point of a tangle's life history. We were able to identify 8103 tau tangles, 132 465 neurons and 299 640 nuclei in tissue samples from three subjects with severe Alzheimer's disease (Braak V-VI) and 4 tau tangles, 200 447 neurons and 462 715 nuclei in tissue samples from three subjects with no significant tau pathology (Braak 0-I). Among these data, we located 57 ghost tangles, which makes them only 0.7% of the total tau tangles observed. We found that ghost tangles are more likely to be found in cortical layers 3 and 5 (49/57), with a select few scattered across other layers 1, 2, 4 and 6. This ability to find rare events, such as ghost tangles, in large enough quantities to statistically test their distribution exemplifies how tissue clearing can be used as a powerful tool for studying selective vulnerability or resilience to pathology across brain regions., Competing Interests: The authors declare no competing interests. Dr. Hyman has a family member who works for Novartis and has equity and serves on the Senior Advisory Boards of Dewpoint and Lattus., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Background: The last few years have seen major advances in blood biomarkers for Alzheimer's Disease (AD) with the development of ultrasensitive immunoassays, promising to transform how we diagnose, prognose, and track progression of neurodegenerative dementias., Methods: We evaluated a panel of four novel ultrasensitive electrochemiluminescence (ECL) immunoassays against presumed CNS derived proteins of interest in AD in plasma [phosphorylated-Tau181 (pTau181), total Tau (tTau), neurofilament light (NfL), and glial fibrillary acidic protein (GFAP)]. Two sets of banked plasma samples from the Massachusetts Alzheimer's Disease Research Center's longitudinal cohort study were examined: A longitudinal prognostic sample ( n = 85) consisting of individuals with mild cognitive impairment (MCI) and 4 years of follow-up and a cross-sectional sample ( n = 238) consisting of individuals with AD, other neurodegenerative diseases (OND), and normal cognition (CN)., Results: Participants with MCI who progressed to dementia due to probable AD during follow-up had higher baseline plasma concentrations of pTau181, NfL, and GFAP compared to non-progressors. The best prognostic discrimination was observed with pTau181 (AUC = 0.83, 1.7-fold increase) and GFAP (AUC = 0.83, 1.6-fold increase). Participants with autopsy- and/or biomarker verified AD had higher plasma levels of pTau181, tTau and GFAP compared to CN and OND, while NfL was elevated in AD and further increased in OND. The best diagnostic discrimination was observed with pTau181 (AD vs CN: AUC = 0.90, 2-fold increase; AD vs. OND: AUC = 0.84, 1.5-fold increase) but tTau, NfL, and GFAP also showed good discrimination between AD and CN (AUC = 0.81-0.85; 1.5-2.2 fold increase)., Conclusions: These new ultrasensitive ECL plasma assays for pTau181, tTau, NfL, and GFAP demonstrated diagnostic utility for detection of AD. Moreover, the absolute baseline plasma levels of pTau181 and GFAP reflect cognitive decline over the next 4 years, providing prognostic information that may have utility in both clinical practice and clinical trial populations., Competing Interests: PK, CC, GS, MS, and SA are named as co-inventors on a US patent application related to neurological biomarker assays that is jointly held by Massachusetts General Hospital and Meso Scale Diagnostics. KJ has served as paid consultant for Bayer, GE Healthcare, Janssen Alzheimer's Immunotherapy, Siemens Medical Solutions, Genzyme, Novartis, Biogen, Roche, ISIS Pharma, AZTherapy, GEHC, Lundberg, and Abbvie. He is a site coinvestigator for Eli Lilly/Avid, Pfizer, Janssen Immunotherapy, and Navidea. He has spoken at symposia sponsored by Janssen Alzheimer's Immunotherapy, and Pfizer. BD has received consulting fees from Acadia, Alector, Arkuda, Biogen, Denali, Eisai, Lilly, Merck, Novartis, Takeda, and Wave Lifesciences, royalties from Cambridge University Press, Elsevier, and Oxford University Press, and research grant support from NIH, Alzheimer's Association, and the Alzheimer's Drug Discovery Foundation. He has been advising for Merck, Wave LifeSciences, Arkuda, Axovant, and Alector. TG-I has received funding from NIH, MassCATS, and Cure Alzhimer's Fund, and served on the Eli Lilly DSMB. DB has received funding from NIA, NINDS, and NIMH, has obtained compensation for serving on the Editorial Board of Belvoir Communications; and is on the Advisory Board of the National Cell Repository for Alzheimer's Disease and the Risk Evaluation Education for Dementia (AGREED) study. BH has received funding from NIH, has served on an Advisory Board for Biogen, and he and/or his family has stock options in Novartis and Dewpoint. AA, PB, CC, GS, and MS are paid employees of Meso Scale Diagnostics, LLC. SA has received honoraria and/or travel expenses for lectures from Abbvie, Biogen, and Eisai, and has served on scientific advisory boards of Corte, has received consulting fees from Abbvie, Boyle Shaughnessy Law, Cognito Therapeutics, Eisai, EIP Pharma, M3 Biotech, Orthogonal Neuroscience, and Risen Pharmaceutical Technology Co., Ltd., and has received research grant support from NIH, Alzheimer's Association, Alzheimer's Drug Discovery Foundation, Challenger Foundation, John Sperling Foundation, Abbvie, Amylyx, Athira Pharma, Chromadex, EIP Pharma, Janssen Pharmaceuticals, Novartis, SEER Biosciences, and vTv Therapeutics, and has served on an MSDB and/or Advisory Board for Allyx Therapeutics, Bob's Last Marathon, Cassava, Cortexyme, Sage Therapeutics, and vTv Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kivisäkk, Carlyle, Sweeney, Trombetta, LaCasse, El-Mufti, Tuncali, Chibnik, Das, Scherzer, Johnson, Dickerson, Gomez-Isla, Blacker, Oakley, Frosch, Hyman, Aghvanyan, Bathala, Campbell, Sigal, Stengelin and Arnold.)

Background and Objectives: We previously found that the APOE genotype affects the rate of cognitive decline in mild-to-moderate Alzheimer disease (AD) dementia independently of its effects on AD neuropathologic changes (ADNC) and copathologies. In this study, we tested the hypothesis that the APOE alleles differentially affect the rate of cognitive decline at the normal aging-early AD continuum and that this association is independent of their effects on classical ADNC and copathologies., Methods: We analyzed APOE associations with the cognitive trajectories (Clinical Dementia Rating scale Sum of Boxes [CDR-SOB] and Mini-Mental State Examination [MMSE]) of more than 1,000 individuals from a national clinicopathologic sample who had either no, mild (sparse neuritic plaques and the Braak neurofibrillary tangle [NFT] stage I/II), or intermediate (moderate neuritic plaques and the Braak NFT stage III/IV) ADNC levels at autopsy via 2 latent classes reverse-time longitudinal modeling., Results: Carrying the APOE ε4 allele was associated with a faster rate of cognitive decline by both CDR-SOB and MMSE relative to APOE ε3 homozygotes. This association remained statistically significant after adjusting for ADNC severity, comorbid pathologies, and the effects of ADNC on the slope of cognitive decline. Our modeling strategy identified 2 latent classes in which APOE ε4 carriers declined faster than APOE ε3 homozygotes, with latent class 1 members representing slow decliners (CDR-SOB: 76.7% of individuals, 0.195 vs 0.146 points/y in APOE ε4 vs APOE ε3/ε3; MMSE: 88.6% of individuals, -0.303 vs -0.153 points/y in APOE ε4 vs APOE ε3/ε3), whereas latent class 2 members were fast decliners (CDR-SOB: 23.3% of participants, 1.536 vs 1.487 points/y in APOE ε4 vs APOE ε3/ε3; MMSE: 11.4% of participants, -2.538 vs -2.387 points/y in APOE ε4 vs APOE ε3/ε3). Compared with slow decliners, fast decliners were more likely to carry the APOE ε4 allele, younger at initial visit and death, more impaired at initial and last visits, and more likely to have intermediate (vs none or mild) ADNC levels, as well as concurrent Lewy bodies and hippocampal sclerosis at autopsy., Discussion: In a large national sample selected to represent the normal aging-early AD continuum, the APOE ε4 allele is associated with a modest but statistically significant acceleration of the cognitive decline rate even after controlling for its effects on ADNC and comorbid pathologies., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Difficulty achieving complete, specific, and homogenous staining is a major bottleneck preventing the widespread use of tissue clearing techniques to image large volumes of human tissue. In this manuscript, we describe a procedure to rapidly design immunostaining protocols for antibody labeling of cleared brain tissue. We prepared libraries of 0.5-1.0 mm thick tissue sections that are fixed, pre-treated, and cleared via similar, but different procedures to optimize staining conditions for a panel of antibodies. Results from a library of mouse tissue correlate well with results from a similarly prepared library of human brain tissue, suggesting mouse tissue is an adequate substitute for protocol optimization. These data show that procedural differences do not influence every antibody-antigen pair in the same way, and minor changes can have deleterious effects, therefore, optimization should be conducted for each target. The approach outlined here will help guide researchers to successfully label a variety of targets, thus removing a major hurdle to accessing the rich 3D information available in large, cleared human tissue volumes., Competing Interests: TZ, RB, ML, BW, AZ, CL, DR No competing interests declared, BH Reviewing editor, eLife, (© 2023, Zwang et al.)

The speed and scope of cognitive deterioration in Alzheimer's disease is highly associated with the advancement of tau neurofibrillary lesions across brain networks. We tested whether the rate of tau propagation is a heritable disease trait in a large, well-characterized cohort of genetically divergent mouse strains. Using an AAV-based model system, P301L-mutant human tau (hTau) was introduced into the entorhinal cortex of mice derived from 18 distinct lines. The extent of tau propagation was measured by distinguishing hTau-producing cells from neurons that were recipients of tau transfer. Heritability calculation revealed that 43% of the variability in tau spread was due to genetic variants segregating across background strains. Strain differences in glial markers were also observed, but did not correlate with tau propagation. Identifying unique genetic variants that influence the progression of pathological tau may uncover novel molecular targets to prevent or slow the pace of tau spread and cognitive decline., Competing Interests: B.T.H. has a family member who works at Novartis and owns stock in Novartis. He serves on the SAB of Dewpoint and owns stock, and he serves on a scientific advisory board or is a consultant for AbbVie, Arvinas, Avrobio, Axon, Biogen, BMS Cell Signaling, Eisai, Genentech, Ionis, Novartis, Sangamo, Takeda, the US Dept of Justice, Vigil, Voyager. His laboratory is supported by Sponsored research agreements with Abbvie, F Prime, and research grants from the National Institutes of Health, Cure Alzheimer’s Fund, Tau Consortium, and the JPB Foundation, and a sponsored research agreement from Abbvie. S.D. is a current employee of Sanofi. A.R.F. is currently affiliated with the Mayo Clinic Graduate School of Biomedical Sciences., (© 2023 The Authors.)

Nucleoporin 98 is a nuclear pore complex component that is mislocalized in Alzheimer's disease and the alteration in nucleoporin 98 has been attributed to tau. In order to determine if nucleoporin 98 mislocalization is a general feature of tauopathies, we assessed the localization of nucleoporin 98 in neurons in primary tauopathies, including frontotemporal lobar degeneration-tau, corticobasal degeneration and progressive supranuclear palsy. Immunofluorescence staining was performed on frontal cortex and occipital cortex tissue from cases of primary tauopathies and controls without neurodegenerative disease using antibodies to identify nucleoporin 98, phospho-tau (Ser202, Thr205) monoclonal antibody and neuronal marker microtubule-associated protein 2. The stained tissue was imaged by fluorescence microscopy and the number of neurons with mislocalized nucleoporin 98 and phospho-tau (Ser202, Thr205) monoclonal antibody staining was quantified. In frontal cortex tissue, all primary tauopathies examined demonstrated significantly increased numbers of neurons with abnormal localization of nucleoporin 98 along the nuclear envelope compared with control tissue. Additionally, frontotemporal lobar degeneration-tau and corticobasal degeneration in the frontal cortex demonstrated significantly increased numbers of neurons with a cytoplasmic mislocalization of nucleoporin 98 compared with control tissue. The number of neurons with mislocalized nucleoporin 98 was significantly correlated with the number of neurons with phospho-tau (Ser202, Thr205) monoclonal antibody-positive tau staining. In the occipital cortex, which is relatively spared from pathological tau accumulations in these primary tauopathies, the localization of nucleoporin 98 was not significantly altered. This study demonstrates that nucleoporin 98 mislocalization is a feature of primary tauopathies and is associated with pathological tau accumulation. In the context of prior research demonstrating nucleoporin 98 mislocalization in Alzheimer's disease and an interaction between tau and nucleoporin 98, these results further support the hypothesis that pathological tau may contribute to nucleoporin 98 mislocalization. Given the critical role of the nuclear pore complex in nucleocytoplasmic transport, the identification of nucleoporin 98 mislocalization in primary tauopathies highlights a potential pathophysiological disruption in these disorders., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)