CSF Biomarkers in Longitudinal Alzheimer Disease Cohorts: Pre-Analytic Challenges.

BACKGROUND: The sensitivity of amyloid to pre-analytic factors complicates cerebrospinal fluid (CSF) diagnostics for Alzheimer disease. We report reliability and validity evidence for automated immunoassays from frozen and fresh CSF samples in an ongoing, single-site research program. METHODS: CSF samples were obtained from 2 Wisconsin cohorts (1256 measurements; 727 participants). Levels of amyloid beta 1-42 (Aß42), phosphorylated tau 181 (pTau181), and total tau (tTau) were obtained using an Elecsys cobas e 601 platform. Repeatability and fixed effects of storage tube type, extraction method, and freezing were assessed via mixed models. Concordance with amyloid positron emission tomography (PET) was investigated with 238 participants having a temporally proximal PET scan. RESULTS: Repeatability was high with intraclass correlation (ICC) ≥0.9, but tube type strongly affected measurements. Discriminative accuracy for PET amyloid positivity was strong across tube types (area under the curve [AUC]: Aß42, 0.87; pTau181Aß42 , 0.96), although optimal thresholds differed. CONCLUSIONS: Under real-world conditions, the Elecsys platform had high repeatability. However, strong effects of pre-analytic factors suggest caution in drawing longitudinal inferences.

Whole genome methylation sequencing in blood identifies extensive differential DNA methylation in late-onset dementia due to Alzheimer's disease.

INTRODUCTION: DNA microarray-based studies report differentially methylated positions (DMPs) in blood between late-onset dementia due to Alzheimer's disease (AD) and cognitively unimpaired individuals, but interrogate < 4% of the genome. METHODS: We used whole genome methylation sequencing (WGMS) to quantify DNA methylation levels at 25,409,826 CpG loci in 281 blood samples from 108 AD and 173 cognitively unimpaired individuals. RESULTS: WGMS identified 28,038 DMPs throughout the human methylome, including 2707 differentially methylated genes (e.g., SORCS3, GABA, and PICALM) encoding proteins in biological pathways relevant to AD such as synaptic membrane, cation channel complex, and glutamatergic synapse. One hundred seventy-three differentially methylated blood-specific enhancers interact with the promoters of 95 genes that are differentially expressed in blood from persons with and without AD. DISCUSSION: WGMS identifies differentially methylated CpGs in known and newly detected genes and enhancers in blood from persons with and without AD. HIGHLIGHTS: Whole genome DNA methylation levels were quantified in blood from persons with and without Alzheimer's disease (AD). Twenty-eight thousand thirty-eight differentially methylated positions (DMPs) were identified. Two thousand seven hundred seven genes comprise DMPs. Forty-eight of 75 independent genetic risk loci for AD have DMPs. One thousand five hundred sixty-eight blood-specific enhancers comprise DMPs, 173 of which interact with the promoters of 95 genes that are differentially expressed in blood from persons with and without AD.

Clinical and Neuropathological Features Associated With Loss of RAB39B.

BACKGROUND: Pathogenic variants in the small GTPase Ras Analogue in Brain 39b (RAB39B) have been linked to the development of early-onset parkinsonism. The study was aimed at delineating the clinical and neuropathological features associated with a previously reported pathogenic variant in RAB39B (c.503C>A p.T168K) and testing for dysregulation of RAB39B in idiopathic PD. METHODS: Clinical details of a male individual hemizygous for the T168K variant were collected by systematic review of medical records. Neuropathological studies of fixed brain tissue were performed and steady-state RAB39B levels were determined by western blot analysis. RESULTS: Neuropathological examination showed extensive dopaminergic neuron loss, widespread Lewy pathology, and iron accumulation in the substantia nigra. Additional pathology was observed in the hippocampus and thalamus. Western blot analysis demonstrated that the T168K variant results in loss of RAB39B. In individuals with idiopathic PD (n = 10, 6 male/4 female), steady-state RAB39B was significantly reduced in the prefrontal cortex and substantia nigra. CONCLUSIONS: T168K RAB39B is unstable in vivo and associated with dopaminergic neuron loss and Lewy pathology. Dysregulation of RAB39B in the prefrontal cortex and substantia nigra of individuals with idiopathic PD potentially implicates the protein more broadly in the pathological mechanisms underlying PD and related Lewy body disorders. © 2020 International Parkinson and Movement Disorder Society.

Nitrous Oxide Impairs Axon Regeneration after Nervous System Injury in Male Rats.

BACKGROUND: Nitrous oxide can induce neurotoxicity. The authors hypothesized that exposure to nitrous oxide impairs axonal regeneration and functional recovery after central nervous system injury. METHODS: The consequences of single and serial in vivo nitrous oxide exposures on axon regeneration in four experimental male rat models of nervous system injury were measured: in vitro axon regeneration in cell culture after in vivo nitrous oxide administration, in vivo axon regeneration after sharp spinal cord injury, in vivo axon regeneration after sharp optic nerve injury, and in vivo functional recovery after blunt contusion spinal cord injury. RESULTS: In vitro axon regeneration 48 h after a single in vivo 70% N2O exposure is less than half that in the absence of nitrous oxide (mean ± SD, 478 ± 275 um; n = 48) versus 210 ± 152 um (n = 48; P < 0.0001). A single exposure to 80% N2O inhibits the beneficial effects of folic acid on in vivo axonal regeneration after sharp spinal cord injury (13.4 ± 7.1% regenerating neurons [n = 12] vs. 0.6 ± 0.7% regenerating neurons [n = 4], P = 0.004). Serial 80% N2O administration reverses the benefit of folic acid on in vivo retinal ganglion cell axon regeneration after sharp optic nerve injury (1277 ± 180 regenerating retinal ganglion cells [n = 7] vs. 895 ± 164 regenerating retinal ganglion cells [n = 7], P = 0.005). Serial 80% N2O exposures reverses the benefit of folic acid on in vivo functional recovery after blunt spinal cord contusion (estimate for fixed effects ± standard error of the estimate: folic acid 5.60 ± 0.54 [n = 9] vs. folic acid + 80% N2O 5.19 ± 0.62 [n = 7], P < 0.0001). CONCLUSIONS: These data indicate that nitrous oxide can impair the ability of central nervous system neurons to regenerate axons after sharp and blunt trauma.

Informed Consent and Cognitive Dysfunction After Noncardiac Surgery in the Elderly.

Cognitive dysfunction 3 months after noncardiac surgery in the elderly satisfies informed consent thresholds of foreseeability in 10%-15% of patients, and materiality with new deficits observed in memory and executive function in patients with normal test performance beforehand. At present, the only safety step to avoid cognitive dysfunction after surgery is to forego surgery, thereby precluding the benefits of surgery with removal of pain and inflammation, and resumption of normal nutrition, physical activity, and sleep. To assure that consent for surgery is properly informed, risks of both cognitive dysfunction and alternative management strategies must be discussed with patients by the surgery team before a procedure is scheduled.

Mutations in RAB39B cause X-linked intellectual disability and early-onset Parkinson disease with α-synuclein pathology.

Advances in understanding the etiology of Parkinson disease have been driven by the identification of causative mutations in families. Genetic analysis of an Australian family with three males displaying clinical features of early-onset parkinsonism and intellectual disability identified a ∼45 kb deletion resulting in the complete loss of RAB39B. We subsequently identified a missense mutation (c.503C>A [p.Thr168Lys]) in RAB39B in an unrelated Wisconsin kindred affected by a similar clinical phenotype. In silico and in vitro studies demonstrated that the mutation destabilized the protein, consistent with loss of function. In vitro small-hairpin-RNA-mediated knockdown of Rab39b resulted in a reduction in the density of α-synuclein immunoreactive puncta in dendritic processes of cultured neurons. In addition, in multiple cell models, we demonstrated that knockdown of Rab39b was associated with reduced steady-state levels of α-synuclein. Post mortem studies demonstrated that loss of RAB39B resulted in pathologically confirmed Parkinson disease. There was extensive dopaminergic neuron loss in the substantia nigra and widespread classic Lewy body pathology. Additional pathological features included cortical Lewy bodies, brain iron accumulation, tau immunoreactivity, and axonal spheroids. Overall, we have shown that loss-of-function mutations in RAB39B cause intellectual disability and pathologically confirmed early-onset Parkinson disease. The loss of RAB39B results in dysregulation of α-synuclein homeostasis and a spectrum of neuropathological features that implicate RAB39B in the pathogenesis of Parkinson disease and potentially other neurodegenerative disorders.

Folate and epigenetic mechanisms in neural tube development and defects.

INTRODUCTION: Multiple genetic and epigenetic factors involved in central nervous system (CNS) development influence the incidence of neural tube defects (NTDs). DISCUSSION: The beneficial effect of periconceptional folic acid on NTD prevention denotes a vital role for the single-carbon biochemical pathway in NTD genesis. Indeed, NTDs are associated with polymorphisms in a diversity of genes that encode folate pathway enzymes. Recent evidence suggests that CNS development and function, and consequently NTDs, are also associated with epigenetic mechanisms, many of which participate in the folate cycle and its input and output pathways. We provide an overview with select examples drawn from the authors' research.

Transgenerational epigenetic inheritance of axonal regeneration after spinal cord injury.

Human epidemiological studies reveal that dietary and environmental alterations influence the health of the offspring and that the effect is not limited to the F1 or F2 generations. Non-Mendelian transgenerational inheritance of traits in response to environmental stimuli has been confirmed in non-mammalian organisms including plants and worms and are shown to be epigenetically mediated. However, transgenerational inheritance beyond the F2 generation remains controversial in mammals. Our lab previously discovered that the treatment of rodents (rats and mice) with folic acid significantly enhances the regeneration of injured axons following spinal cord injury in vivo and in vitro, and the effect is mediated by DNA methylation. The potential heritability of DNA methylation prompted us to investigate the following question: Is the enhanced axonal regeneration phenotype inherited transgenerationally without exposure to folic acid supplementation in the intervening generations? In the present review, we condense our findings showing that a beneficial trait (i.e., enhanced axonal regeneration after spinal cord injury) and accompanying molecular alterations (i.e., DNA methylation), triggered by an environmental exposure (i.e., folic acid supplementation) to F0 animals only, are inherited transgenerationally and beyond the F3 generation.

Plasma phosphorylated tau 217 in preclinical Alzheimer's disease.

An accurate blood test for Alzheimer's disease that is sensitive to preclinical proteinopathy and cognitive decline has clear implications for early detection and secondary prevention. We assessed the performance of plasma phosphorylated tau 217 ( pTa u 217 ) against brain PET markers of amyloid [ [ 11 C ] -labelled Pittsburgh compound B (PiB)] and tau ( [ 18 F ] MK-6240) and its utility for predicting longitudinal cognition. Samples were analysed from a subset of participants with up to 8 years follow-up in the Wisconsin Registry for Alzheimer's Prevention (WRAP; 2001-present; plasma 2011-present), a longitudinal cohort study of adults from midlife, enriched for parental history of Alzheimer's disease. Participants were a convenience sample who volunteered for at least one PiB scan, had usable banked plasma and were cognitively unimpaired at first plasma collection. Study personnel who interacted with participants or samples were blind to amyloid status. We used mixed effects models and receiver-operator characteristic curves to assess concordance between plasma pTa u 217 and PET biomarkers of Alzheimer's disease and mixed effects models to understand the ability of plasma pTa u 217 to predict longitudinal performance on WRAP's preclinical Alzheimer's cognitive composite (PACC-3). The primary analysis included 165 people (108 women; mean age = 62.9 ± 6.06; 160 still enrolled; 2 deceased; 3 discontinued). Plasma pTa u 217 was strongly related to PET-based estimates of concurrent brain amyloid ( ß ^ = 0.83 (0.75, 0.90), P < 0.001). Concordance was high between plasma pTa u 217 and both amyloid PET (area under the curve = 0.91, specificity = 0.80, sensitivity = 0.85, positive predictive value = 0.58, negative predictive value = 0.94) and tau PET (area under the curve = 0.95, specificity = 1, sensitivity = 0.85, positive predictive value = 1, negative predictive value = 0.98). Higher baseline pTa u 217 levels were associated with worse cognitive trajectories ( ß ^ p T a u × a g e = -0.07 (-0.09, -0.06), P < 0.001). In a convenience sample of unimpaired adults, plasma pTa u 217 levels correlate well with concurrent brain Alzheimer's disease pathophysiology and with prospective cognitive performance. These data indicate that this marker can detect disease before clinical signs and thus may disambiguate presymptomatic Alzheimer's disease from normal cognitive ageing.

Persistent blood DNA methylation changes one year after SARS-CoV-2 infection.

We recently reported the COVID-19-induced circulating leukocytes DNA methylation profile. Here, we hypothesized that some of these genes would persist differentially methylated after disease resolution. Fifteen participants previously hospitalized for SARS-CoV-2 infection were epityped one year after discharge. Of the 1505 acute illness-induced differentially methylated regions (DMRs) previously identified, we found 71 regions with persisted differentially methylated, with an average of 7 serial CpG positions per DMR. Sixty-four DMRs persisted hypermethylated, and 7 DMR persisted hypomethylated. These data are the first reported evidence that DNA methylation changes in circulating leukocytes endure long after recovery from acute illness.

The effect of TOMM40 poly-T length on gray matter volume and cognition in middle-aged persons with APOE ε3/ε3 genotype.

OBJECTIVE: Apolipoprotein E (APOE) genotypes are associated with variable risk of developing late-onset Alzheimer's disease (LOAD), with APOE epsilon 4 (APOE ε4) having higher risk. A variable poly-T length polymorphism at rs10524523, within intron 6 of the translocase of the outer mitochondrial membrane (TOMM40) gene, has been shown to influence age of onset in LOAD, with very long (VL) poly-T length associated with earlier disease onset, and short poly-T length associated with later onset. In this study, we tested the hypothesis that brain and cognitive changes suggestive of presymptomatic LOAD may be associated with this TOMM40 polymorphism. METHODS: Among healthy APOE ε3 homozygous adults (N = 117; mean age, 55 years), we compared those who were homozygous for VL/VL (n = 35) TOMM40 poly-T lengths (who were presumably at higher risk) with those homozygous for short (S/S; n = 38) poly-T lengths, as well as those with heterozygous (S/VL; n = 44) poly-T length polymorphisms, on measures of learning and memory and on structural brain imaging. RESULTS: The VL/VL group showed lower performance than the S/S TOMM40 group on primacy retrieval from a verbal list learning task, a finding which is also seen in early Alzheimer's disease. A dose-dependent increase in the VL TOMM40 polymorphism (from no VL alleles, to S/VL heterozygous, to VL/VL homozygous) was associated with decreasing gray matter volume in the ventral posterior cingulate and medial ventral precuneus, a region of the brain affected early in LOAD. CONCLUSIONS: These findings among APOE ε3/ε3 late middle-aged adults suggest that a subgroup with VL TOMM40 poly-T lengths may be experiencing incipient LOAD-related cognitive and brain changes.

DNA methylation and hydroxymethylation have distinct genome-wide profiles related to axonal regeneration.

Alterations in environmentally sensitive epigenetic mechanisms (e.g., DNA methylation) influence axonal regeneration in the spinal cord following sharp injury. Conventional DNA methylation detection methods using sodium bisulphite treatment do not distinguish between methylated and hydroxymethylated forms of cytosine, meaning that past studies report a composite of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). To identify the distinct contributions of DNA methylation modifications to axonal regeneration, we collected spinal cord tissue after sharp injury from untreated adult F3 male rats with enhanced regeneration of injured spinal axons or controls, derived from folate- or water-treated F0 lineages, respectively. Genomic DNA was profiled for genome-wide 5hmC levels, revealing 658 differentially hydroxymethylated regions (DhMRs). Genomic profiling with whole genome bisulphite sequencing disclosed regeneration-related alterations in composite 5mC + 5hmC DNA methylation levels at 2,260 differentially methylated regions (DMRs). While pathway analyses revealed that differentially hydroxymethylated and methylated genes are linked to biologically relevant axon developmental pathways, only 22 genes harbour both DhMR and DMRs. Since these differential modifications were more than 60 kilobases on average away from each other, the large majority of differential hydroxymethylated and methylated regions are unique with distinct functions in the axonal regeneration phenotype. These data highlight the importance of distinguishing independent contributions of 5mC and 5hmC levels in the central nervous system, and denote discrete roles for DNA methylation modifications in spinal cord injury and regeneration in the context of transgenerational inheritance.

Blood DNA methylation and COVID-19 outcomes.

BACKGROUND: There are no prior reports that compare differentially methylated regions of DNA in blood samples from COVID-19 patients to samples collected before the SARS-CoV-2 pandemic using a shared epigenotyping platform. We performed a genome-wide analysis of circulating blood DNA CpG methylation using the Infinium Human MethylationEPIC BeadChip on 124 blood samples from hospitalized COVID-19-positive and COVID-19-negative patients and compared these data with previously reported data from 39 healthy individuals collected before the pandemic. Prospective outcome measures such as COVID-19-GRAM risk-score and mortality were combined with methylation data. RESULTS: Global mean methylation levels did not differ between COVID-19 patients and healthy pre-pandemic controls. About 75% of acute illness-associated differentially methylated regions were located near gene promoter regions and were hypo-methylated in comparison with healthy pre-pandemic controls. Gene ontology analyses revealed terms associated with the immune response to viral infections and leukocyte activation; and disease ontology analyses revealed a predominance of autoimmune disorders. Among COVID-19-positive patients, worse outcomes were associated with a prevailing hyper-methylated status. Recursive feature elimination identified 77 differentially methylated positions predictive of COVID-19 severity measured by the GRAM-risk score. CONCLUSION: Our data contribute to the awareness that DNA methylation may influence the expression of genes that regulate COVID-19 progression and represent a targetable process in that setting.

Metabolites Associated with Early Cognitive Changes Implicated in Alzheimer's Disease.

BACKGROUND: Understanding metabolic mechanisms associated with cognitive changes preceding an Alzheimer's disease (AD) diagnosis could advance our understanding of AD progression and inform preventive methods. OBJECTIVE: We investigated the metabolomics of the early changes in executive function and delayed recall, the earliest aspects of cognitive function to change in the course of AD development, in order to better understand mechanisms that could contribute to early stages and progression of this disease. METHODS: This investigation used longitudinal plasma samples from the Wisconsin Registry for Alzheimer's Prevention (WRAP), a cohort of participants who were dementia free at enrollment and enriched with a parental history of AD. Metabolomic profiles were quantified for 2,324 fasting plasma samples among 1,200 participants, each with up to three study visits, which occurred every two years. Metabolites were individually tested for association with executive function and delayed recall trajectories across age. RESULTS: Of 1,097 metabolites tested, levels of seven were associated with executive function trajectories, including an amino acid cysteine S-sulfate and three fatty acids, including erucate (22 : 1n9), while none were associated with delayed recall trajectories. Replication was attempted for four of these metabolites that were present in the Vietnam Era Twin Study of Aging (VETSA). Although none reached statistical significance, three of these associations showed consistent effectdirections. CONCLUSION: Our results suggest potential metabolomic mechanisms that could contribute to the earliest signs of cognitive decline. In particular, fatty acids may be associated with cognition in a manner that is more complex than previously suspected.