Protocol for hybrid structured-light three-dimensional scanning and modeling of human postmortem brains.

Despite numerous neuropathological criteria for the evaluation of microscopic tissue slides, there are no systemic standards for sectioning postmortem human brain tissue. Here, we present a protocol for postmortem brain hybrid structured-light scanning to facilitate 3D printing of sectioning matrices. We describe steps for tissue preparation, 3D scanning and printing, and matrix-guided sectioning. This protocol can be used for streamlining tissue histopathology as well as in brain morphology volumetric analyses and applications in medical education. For complete details on the use and execution of this protocol, please refer to Barannikov et al.1.

Upper motor neuron-predominant motor neuron disease presenting as atypical parkinsonism: A clinicopathological study.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by upper and lower motor neuron signs. There are, however, cases where upper motor neurons (UMNs) are predominantly affected, leading to clinical presentations of UMN-dominant ALS or primary lateral sclerosis. Furthermore, cases exhibiting an UMN-predominant pattern of motor neuron disease (MND) presenting with corticobasal syndrome (CBS) have been sparsely reported. This study aims to clarify the clinicopathological features of patients with UMN-predominant MND. We reviewed 24 patients with UMN-predominant MND with TDP-43 pathology in the presence or absence of frontotemporal lobar degeneration. Additionally, we reviewed the medical records of patients with pathologically-confirmed corticobasal degeneration (CBD) who received a final clinical diagnosis of CBS (n = 10) and patients with pathologically-confirmed progressive supranuclear palsy (PSP) who received a final clinical diagnosis of PSP syndrome (n = 10). Of 24 UMN-predominant MND patients, 20 had a clinical diagnosis of an atypical parkinsonian disorder, including CBS (n = 11) and PSP syndrome (n = 8). Only two patients had antemortem diagnoses of motor neuron disease. UMN-predominant MND patients with CBS less frequently exhibited apraxia than those with CBD, and they were less likely to meet clinical criteria for possible or probable CBS. Similarly, UMN-predominant MND patients with PSP syndrome less often met clinical criteria for probable PSP than PSP patients with PSP syndrome. Our findings suggest that UMN-predominant MND can mimic atypical parkinsonism, and should be considered in the differential diagnosis of CBS and PSP syndrome, in particular when criteria are not met.

Deciphering Distinct Genetic Risk Factors for FTLD-TDP Pathological Subtypes via Whole-Genome Sequencing.

Frontotemporal lobar degeneration with neuronal inclusions of the TAR DNA-binding protein 43 (FTLD-TDP) is a fatal neurodegenerative disorder with only a limited number of risk loci identified. We report our comprehensive genome-wide association study as part of the International FTLD-TDP Whole-Genome Sequencing Consortium, including 985 cases and 3,153 controls, and meta-analysis with the Dementia-seq cohort, compiled from 26 institutions/brain banks in the United States, Europe and Australia. We confirm UNC13A as the strongest overall FTLD-TDP risk factor and identify TNIP1 as a novel FTLD-TDP risk factor. In subgroup analyses, we further identify for the first time genome-wide significant loci specific to each of the three main FTLD-TDP pathological subtypes (A, B and C), as well as enrichment of risk loci in distinct tissues, brain regions, and neuronal subtypes, suggesting distinct disease aetiologies in each of the subtypes. Rare variant analysis confirmed TBK1 and identified VIPR1 , RBPJL , and L3MBTL1 as novel subtype specific FTLD-TDP risk genes, further highlighting the role of innate and adaptive immunity and notch signalling pathway in FTLD-TDP, with potential diagnostic and novel therapeutic implications.

Distinct patterns of plaque and microglia glycosylation in Alzheimer's disease.

Glycosylation is the most common form of post-translational modification in the brain. Aberrant glycosylation has been observed in cerebrospinal fluid and brain tissue of Alzheimer's disease (AD) cases, including dysregulation of terminal sialic acid (SA) modifications. While alterations in sialylation have been identified in AD, the localization of SA modifications on cellular or aggregate-associated glycans is largely unknown because of limited spatial resolution of commonly utilized methods. The present study aims to overcome these limitations with novel combinations of histologic techniques to characterize the sialylation landscape of O- and N-linked glycans in autopsy-confirmed AD post-mortem brain tissue. Sialylated glycans facilitate important cellular functions including cell-to-cell interaction, cell migration, cell adhesion, immune regulation, and membrane excitability. Previous studies have not investigated both N- and O-linked sialylated glycans in neurodegeneration. In this study, the location and distribution of sialylated glycans were evaluated in three brain regions (frontal cortex, hippocampus, and cerebellum) from 10 AD cases using quantitative digital pathology techniques. Notably, we found significantly greater N-sialylation of the Aß plaque microenvironment compared with O-sialylation. Plaque-associated microglia displayed the most intense N-sialylation proximal to plaque pathology. Further analyses revealed distinct differences in the levels of N- and O-sialylation between cored and diffuse Aß plaque morphologies. Interestingly, phosphorylated tau pathology led to a slight increase in N-sialylation and no influence of O-sialylation in these AD brains. Confirming our previous observations in mice with novel histologic approach, these findings support microglia sialylation appears to have a relationship with AD protein aggregates while providing potential targets for therapeutic strategies.

Quinolinic acid links kidney injury to brain toxicity.

Kidney dysfunction often leads to neurological impairment, yet the complex kidney-brain relationship remains elusive. We employed spatial and bulk metabolomics to investigate a mouse model of rapid kidney failure induced by mouse double minute 2 ( Mdm2) conditional deletion in the kidney tubules to interrogate kidney and brain metabolism. Pathway enrichment analysis of focused plasma metabolomics panel pinpointed tryptophan metabolism as the most altered pathway with kidney failure. Spatial metabolomics showed toxic tryptophan metabolites in the kidneys and brains, revealing a novel connection between advanced kidney disease and accelerated kynurenine degradation. In particular, the excitotoxic metabolite quinolinic acid was localized in ependymal cells adjacent to the ventricle in the setting of kidney failure. These findings were associated with brain inflammation and cell death. A separate mouse model of acute kidney injury also had an increase in circulating toxic tryptophan metabolites along with altered brain inflammation. Patients with advanced CKD similarly demonstrated elevated plasma kynurenine metabolites and quinolinic acid was uniquely correlated with fatigue and reduced quality of life in humans. Overall, our study identifies the kynurenine pathway as a bridge between kidney decline, systemic inflammation, and brain toxicity, offering potential avenues for diagnosis and treatment of neurological issues in kidney disease.

Nanopore-based DNA long-read sequencing analysis of the aged human brain.

Aging disrupts cellular processes such as DNA repair and epigenetic control, leading to a gradual buildup of genomic alterations that can have detrimental effects in post-mitotic cells. Genomic alterations in regions of the genome that are rich in repetitive sequences, often termed "dark loci," are difficult to resolve using traditional sequencing approaches. New long-read technologies offer promising avenues for exploration of previously inaccessible regions of the genome. Using nanopore-based long-read whole-genome sequencing of DNA extracted from aged 18 human brains, we identify previously unreported structural variants and methylation patterns within repetitive DNA, focusing on transposable elements ("jumping genes") as crucial sources of variation, particularly in dark loci. Our analyses reveal potential somatic insertion variants and provides DNA methylation frequencies for many retrotransposon families. We further demonstrate the utility of this technology for the study of these challenging genomic regions in brains affected by Alzheimer's disease and identify significant differences in DNA methylation in pathologically normal brains versus those affected by Alzheimer's disease. Highlighting the power of this approach, we discover specific polymorphic retrotransposons with altered DNA methylation patterns. These retrotransposon loci have the potential to contribute to pathology, warranting further investigation in Alzheimer's disease research. Taken together, our study provides the first long-read DNA sequencing-based analysis of retrotransposon sequences, structural variants, and DNA methylation in the aging brain affected with Alzheimer's disease neuropathology.

Multi-omics analyses reveal novel effects of PLCγ2 deficiency in the mouse brain.

Phospholipase C gamma-2 (PLCγ2) catalyzes the hydrolysis of the membrane phosphatidylinositol-4,5-bisphosphate (PIP2) to form diacylglycerol (DAG) and inositol trisphosphate (IP3), which subsequently feed into numerous downstream signaling pathways. PLCG2 polymorphisms are associated with both reduced and increased risk of Alzheimer's disease (AD) and with longevity. In the brain, PLCG2 is highly expressed in microglia, where it is proposed to regulate phagocytosis, secretion of cytokines/chemokines, cell survival and proliferation. We analyzed the brains of three-month-old PLCγ2 knockout (KO), heterozygous (HET), and wild-type (WT) mice using multiomics approaches, including shotgun lipidomics, proteomics, and gene expression profiling, and immunofluorescence. Lipidomic analyses revealed sex-specific losses of total cerebrum PIP2 and decreasing trends of DAG content in KOs. In addition, PLCγ2 depletion led to significant losses of myelin-specific lipids and decreasing trends of myelin-enriched lipids. Consistent with our lipidomics results, RNA profiling revealed sex-specific changes in the expression levels of several myelin-related genes. Further, consistent with the available literature, gene expression profiling revealed subtle changes on microglia phenotype in mature adult KOs under baseline conditions, suggestive of reduced microglia reactivity. Immunohistochemistry confirmed subtle differences in density of microglia and oligodendrocytes in KOs. Exploratory proteomic pathway analyses revealed changes in KO and HET females compared to WTs, with over-abundant proteins pointing to mTOR signaling, and under-abundant proteins to oligodendrocytes. Overall, our data indicate that loss of PLCγ2 has subtle effects on brain homeostasis that may underlie enhanced vulnerability to AD pathology and aging via novel mechanisms in addition to regulation of microglia function.

Central nervous system sulfatide deficiency as a causal factor for bladder disorder in Alzheimer's disease.

BACKGROUND: Despite being a brain disorder, Alzheimer's disease (AD) is often accompanied by peripheral organ dysregulations (e.g., loss of bladder control in late-stage AD), which highly rely on spinal cord coordination. However, the causal factor(s) for peripheral organ dysregulation in AD remain elusive. METHODS: The central nervous system (CNS) is enriched in lipids. We applied quantitative shotgun lipidomics to determine lipid profiles of human AD spinal cord tissues. Additionally, a CNS sulfatide (ST)-deficient mouse model was used to study the lipidome, transcriptome and peripheral organ phenotypes of ST loss. RESULTS: We observed marked myelin lipid reduction in the spinal cord of AD subjects versus cognitively normal individuals. Among which, levels of ST, a myelin-enriched lipid class, were strongly and negatively associated with the severity of AD. A CNS myelin-specific ST-deficient mouse model was used to further identify the causes and consequences of spinal cord lipidome changes. Interestingly, ST deficiency led to spinal cord lipidome and transcriptome profiles highly resembling those observed in AD, characterized by decline of multiple myelin-enriched lipid classes and enhanced inflammatory responses, respectively. These changes significantly disrupted spinal cord function and led to substantial enlargement of urinary bladder in ST-deficient mice. CONCLUSIONS: Our study identified CNS ST deficiency as a causal factor for AD-like lipid dysregulation, inflammation response and ultimately the development of bladder disorders. Targeting to maintain ST levels may serve as a promising strategy for the prevention and treatment of AD-related peripheral disorders.

Moesin is an effector of tau-induced actin overstabilization, cell cycle activation, and neurotoxicity in Alzheimer's disease.

In Alzheimer's disease, neurons acquire phenotypes that are also present in various cancers, including aberrant activation of the cell cycle. Unlike cancer, cell cycle activation in post-mitotic neurons is sufficient to induce cell death. Multiple lines of evidence suggest that abortive cell cycle activation is a consequence of pathogenic forms of tau, a protein that drives neurodegeneration in Alzheimer's disease and related "tauopathies." Here we combine network analyses of human Alzheimer's disease and mouse models of Alzheimer's disease and primary tauopathy with studies in Drosophila to discover that pathogenic forms of tau drive cell cycle activation by disrupting a cellular program involved in cancer and the epithelial-mesenchymal transition (EMT). Moesin, an EMT driver, is elevated in cells harboring disease-associated phosphotau, over-stabilized actin, and ectopic cell cycle activation. We further find that genetic manipulation of Moesin mediates tau-induced neurodegeneration. Taken together, our study identifies novel parallels between tauopathy and cancer.

Increased α-2,6 sialic acid on microglia in amyloid pathology is resistant to oseltamivir.

Terminal sialic acid residues are present on most glycoproteins and glycolipids, but levels of sialylation are known to change in the brain throughout the lifespan as well as during disease. Sialic acids are important for numerous cellular processes including cell adhesion, neurodevelopment, and immune regulation as well as pathogen invasion into host cells. Neuraminidase enzymes, also known as sialidases, are responsible for removal of terminal sialic acids in a process known as desialylation. Neuraminidase 1 (Neu1) cleaves the α-2,6 bond of terminal sialic acids. Aging individuals with dementia are often treated with the antiviral medication oseltamivir, which is associated with induction of adverse neuropsychiatric side effects; this drug inhibits both viral and mammalian Neu1. The present study tested whether a clinically relevant antiviral dosing regimen of oseltamivir would disrupt behavior in the 5XFAD mouse model of Alzheimer's disease amyloid pathology or wild-type littermates. While oseltamivir treatment did not impact mouse behavior or modify amyloid plaque size or morphology, a novel spatial distribution of α-2,6 sialic acid residues was discovered in 5XFAD mice that was not present in wild-type littermates. Further analyses revealed that α-2,6 sialic acid residues were not localized the amyloid plaques but instead localized to plaque-associated microglia. Notably, treatment with oseltamivir did not alter α-2,6 sialic acid distribution on plaque-associated microglia in 5XFAD mice which may be due to downregulation of Neu1 transcript levels in 5XFAD mice. Overall, this study suggests that plaque-associated microglia are highly sialylated and are resistant to change with oseltamivir, thus interfering with microglia immune recognition of and response to amyloid pathology.

Pathogenic tau-induced transposable element-derived dsRNA drives neuroinflammation.

Deposition of tau protein aggregates in the brain of affected individuals is a defining feature of "tauopathies," including Alzheimer's disease. Studies of human brain tissue and various model systems of tauopathy report that toxic forms of tau negatively affect nuclear and genomic architecture, identifying pathogenic tau-induced heterochromatin decondensation and consequent retrotransposon activation as a causal mediator of neurodegeneration. On the basis of their similarity to retroviruses, retrotransposons drive neuroinflammation via toxic intermediates, including double-stranded RNA (dsRNA). We find that dsRNA and dsRNA sensing machinery are elevated in astrocytes of postmortem brain tissue from patients with Alzheimer's disease and progressive supranuclear palsy and in brains of tau transgenic mice. Using a Drosophila model of tauopathy, we identify specific tau-induced retrotransposons that form dsRNA and find that pathogenic tau and heterochromatin decondensation causally drive dsRNA-mediated neurodegeneration and neuroinflammation. Our study suggests that pathogenic tau-induced heterochromatin decondensation and retrotransposon activation cause elevation of inflammatory, transposable element-derived dsRNA in the adult brain.

Neuroimmune proteins can differentiate between tauopathies.

BACKGROUND: Tauopathies are a group of neurodegenerative diseases where there is pathologic accumulation of hyperphosphorylated tau protein (ptau). The most common tauopathy is Alzheimer's disease (AD), but chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and argyrophilic grain disease (AGD) are significant health risks as well. Currently, it is unclear what specific molecular factors might drive each distinct disease and represent therapeutic targets. Additionally, there is a lack of biomarkers that can differentiate each disease in life. Recent work has suggested that neuroinflammatory changes might be specific among distinct diseases and offers a novel resource for mechanistic targets and biomarker candidates. METHODS: To better examine each tauopathy, a 71 immune-related protein multiplex ELISA panel was utilized to analyze anterior cingulate grey matter from 127 individuals neuropathologically diagnosed with AD, CTE, PSP, CBD, and AGD. A partial least square regression analysis was carried out to perform unbiased clustering and identify proteins that are distinctly correlated with each tauopathy correcting for age and gender. Receiver operator characteristic and binary logistic regression analyses were then used to examine the ability of each candidate protein to distinguish diseases. Validation in postmortem cerebrospinal fluid (CSF) from 15 AD and 14 CTE cases was performed to determine if candidate proteins could act as possible novel biomarkers. RESULTS: Five clusters of immune proteins were identified and compared to each tauopathy to determine if clusters were specific to distinct disease. Each cluster was found to correlate with either CTE, AD, PSP, CBD, or AGD. When examining which proteins were the strongest driver of each cluster, it was observed the most distinctive protein for CTE was CCL21, AD was FLT3L, and PSP was IL13. Individual proteins that were specific to CBD and AGD were not observed. CCL21 was observed to be elevated in CTE CSF compared to AD cases (p = 0.02), further validating the use as possible biomarkers. Sub-analyses for male only cases confirmed the results were not skewed by gender differences. CONCLUSIONS: Overall, these results highlight that different neuroinflammatory responses might underlie unique mechanisms in related neurodegenerative pathologies. Additionally, the use of distinct neuroinflammatory signatures could help differentiate between tauopathies and act as novel biomarker candidate to increase specificity for in-life diagnoses.

Differential Vulnerability of Hippocampal Subfields in Primary Age-Related Tauopathy and Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a tauopathy associated with repetitive mild head impacts characterized by perivascular hyperphosphorylated tau (p-tau) in neurofibrillary tangles (NFTs) and neurites in the depths of the neocortical sulci. In moderate to advanced CTE, NFTs accumulate in the hippocampus, potentially overlapping neuroanatomically with primary age-related tauopathy (PART), an age-related tauopathy characterized by Alzheimer disease-like tau pathology in the hippocampus devoid of amyloid plaques. We measured p-tau burden using positive-pixel counts on immunohistochemically stained and neuroanatomically segmented hippocampal tissue. Subjects with CTE had a higher total p-tau burden than PART subjects in all sectors (p = 0.005). Within groups, PART had significantly higher total p-tau burden in CA1/subiculum compared to CA3 (p = 0.02) and CA4 (p = 0.01) and total p-tau burden in CA2 trended higher than CA4 (p = 0.06). In CTE, total p-tau burden in CA1/subiculum was significantly higher than in the dentate gyrus; and CA2 also trended higher than dentate gyrus (p = 0.01, p = 0.06). When controlling for p-tau burden across the entire hippocampus, CA3 and CA4 had significantly higher p-tau burden in CTE than PART (p < 0.0001). These data demonstrate differences in hippocampal p-tau burden and regional distribution in CTE compared to PART that might be helpful in differential diagnosis and reveal insights into disease pathogenesis.

Microglia: Friend and foe in tauopathy.

Aggregation of misfolded microtubule associated protein tau into abnormal intracellular inclusions defines a class of neurodegenerative diseases known as tauopathies. The consistent spatiotemporal progression of tau pathology in Alzheimer's disease (AD) led to the hypothesis that tau aggregates spread in the brain via bioactive tau "seeds" underlying advancing disease course. Recent studies implicate microglia, the resident immune cells of the central nervous system, in both negative and positive regulation of tau pathology. Polymorphisms in genes that alter microglial function are associated with the development of AD and other tauopathies. Experimental manipulation of microglia function can alter tau pathology and microglia-mediated neuroinflammatory cascades can exacerbate tau pathology. Microglia also exert protective functions by mitigating tau spread: microglia internalize tau seeds and have the capacity to degrade them. However, when microglia fail to degrade these tau seeds there are deleterious consequences, including secretion of exosomes containing tau that can spread to neurons. This review explores the intersection of microglia and tau from the perspective of neuropathology, neuroimaging, genetics, transcriptomics, and molecular biology. As tau-targeted therapies such as anti-tau antibodies advance through clinical trials, it is critical to understand the interaction between tau and microglia.

Asymmetry of Hippocampal Tau Pathology in Primary Age-Related Tauopathy and Alzheimer Disease.

Primary age-related tauopathy (PART) is a neurodegenerative entity defined as neurofibrillary degeneration generally restricted to the medial temporal region (Braak stage I-IV) with complete or near absence of diffuse and neuritic plaques. Symptoms range in severity but are generally milder and later in onset than in Alzheimer disease (AD). Recently, an early predilection for neurofibrillary degeneration in the hippocampal CA2 subregion has been demonstrated in PART, whereas AD neuropathologic change (ADNC) typically displays relative sparing of CA2 until later stages. In this study, we utilized a semiquantitative scoring system to evaluate asymmetry of neurofibrillary degeneration between left and right hippocampi in 67 PART cases and 17 ADNC cases. 49% of PART cases demonstrated asymmetric findings in at least one hippocampal subregion, and 79% of the asymmetric cases displayed some degree of CA2 asymmetry. Additionally, 19% of cases revealed a difference in Braak score between the right and left hippocampi. There was a significant difference in CA2 neurofibrillary degeneration (p = 0.0006) and CA2/CA1 ratio (p < 0.0001) when comparing the contralateral sides, but neither right nor left was more consistently affected. These data show the importance of analyzing bilateral hippocampi in the diagnostic evaluation of PART and potentially of other neurodegenerative diseases.

The Second NINDS/NIBIB Consensus Meeting to Define Neuropathological Criteria for the Diagnosis of Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disorder associated with exposure to head trauma. In 2015, a panel of neuropathologists funded by the NINDS/NIBIB defined preliminary consensus neuropathological criteria for CTE, including the pathognomonic lesion of CTE as "an accumulation of abnormal hyperphosphorylated tau (p-tau) in neurons and astroglia distributed around small blood vessels at the depths of cortical sulci and in an irregular pattern," based on review of 25 tauopathy cases. In 2016, the consensus panel met again to review and refine the preliminary criteria, with consideration around the minimum threshold for diagnosis and the reproducibility of a proposed pathological staging scheme. Eight neuropathologists evaluated 27 cases of tauopathies (17 CTE cases), blinded to clinical and demographic information. Generalized estimating equation analyses showed a statistically significant association between the raters and CTE diagnosis for both the blinded (OR = 72.11, 95% CI = 19.5-267.0) and unblinded rounds (OR = 256.91, 95% CI = 63.6-1558.6). Based on the challenges in assigning CTE stage, the panel proposed a working protocol including a minimum threshold for CTE diagnosis and an algorithm for the assessment of CTE severity as "Low CTE" or "High CTE" for use in future clinical, pathological, and molecular studies.