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.

Changes in vitamin D metabolites at the time of critical illness and 6 months later-A prospective observational study.

BACKGROUND: Despite multiple studies suggesting that low 25(OH)D-vitamin levels are associated with worse outcomes in critically ill individuals, attempts to mitigate the outcomes by fixed dose enteral supplementation unguided by baseline or target blood levels have been unsuccessful. Since a single measurement of 25(OH)D may not optimally reflect an individual's vitamin D status, we studied the plasma concentration of different vitamin D metabolites and their recovery during and following resolution of acute critical illness. METHODS: A prospective observational study including patients 18 years and older admitted to a mixed medical-surgical ICU in Reykjavik, Iceland, located at a high-northern altitude (64° N). Vitamin D metabolites were measured at three timepoints; On admission (S1), 3-5 days following admission (S2) and after recovery from acute illness (median 178 days) (S3). Concentrations of total 25(OH)D-vitamin, cholecalciferol (D3 ), total 24,25(OH)D-vitamin, vitamin D binding protein (VDBP) were measured with LC-tandem mass spectrometry (LC-MS/MS) and free 25-(OH)D was measured with enzyme-linked immunosorbent assay. RESULTS: Most individuals were vitamin D deficient when assessed during critical illness, with 25(OH)D-vitamin levels under 30 ng/ml for 37/40 individuals at timepoint S1 and 34/38 at S2. After recovery, 18/30 patients were deficient at S3. Levels of all vitamin D metabolites measured were low during critical illness but rose substantially following resolution of acute illness. No strong correlation was found between markers of acute illness severity or duration and resolution of vitamin D metabolites in the interval between acute illness and recovery. CONCLUSIONS: In critically ill patients, levels of multiple vitamin D metabolites are low but substantial recovery occurs following resolution of acute illness. It is unclear whether a single metabolite is sufficient to assess vitamin D status of critically ill patients and guide potential supplementation.

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.

Lamin B1 duplications cause autosomal dominant leukodystrophy.

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by symmetrical widespread myelin loss in the central nervous system, with a phenotype similar to chronic progressive multiple sclerosis. In this study, we identify a genomic duplication that causes ADLD. Affected individuals carry an extra copy of the gene for the nuclear laminar protein lamin B1, resulting in increased gene dosage in brain tissue from individuals with ADLD. Increased expression of lamin B1 in Drosophila melanogaster resulted in a degenerative phenotype. In addition, an abnormal nuclear morphology was apparent when cultured cells overexpressed this protein. This is the first human disease attributable to mutations in the gene encoding lamin B1. Antibodies to lamin B are found in individuals with autoimmune diseases, and it is also an antigen recognized by a monoclonal antibody raised against plaques from brains of individuals with multiple sclerosis. This raises the possibility that lamin B may be a link to the autoimmune attack that occurs in multiple sclerosis.

Folate-mediated transgenerational inheritance of sperm DNA methylation patterns correlate with spinal axon regeneration.

In mammals, the molecular mechanisms underlying transgenerational inheritance of phenotypic traits in serial generations of progeny after ancestral environmental exposures, without variation in DNA sequence, remain elusive. We've recently described transmission of a beneficial trait in rats and mice, in which F0 supplementation of methyl donors, including folic acid, generates enhanced axon regeneration after sharp spinal cord injury in untreated F1 to F3 progeny linked to differential DNA methylation levels in spinal cord tissue. To test whether the transgenerational effect of folic acid is transmitted via the germline, we performed whole-genome methylation sequencing on sperm DNA from F0 mice treated with either folic acid or vehicle control, and their F1, F2, and F3 untreated progeny. Transgenerational differentially methylated regions (DMRs) are observed in each consecutive generation and distinguish folic acid from untreated lineages, predominate outside of CpG islands and in regions of the genome that regulate gene expression, including promoters, and overlap at both the differentially methylated position (DMP) and gene levels. These findings indicate that molecular changes between generations are caused by ancestral folate supplementation. In addition, 29,719 DMPs exhibit serial increases or decreases in DNA methylation levels in successive generations of untreated offspring, correlating with a serial increase in the phenotype across generations, consistent with a 'wash-in' effect. Sibship-specific DMPs annotate to genes that participate in axon- and synapse-related pathways.

Whole genome methylation sequencing in blood from persons with mild cognitive impairment and dementia due to Alzheimer's disease identifies cognitive status.

INTRODUCTION: Whole genome methylation sequencing (WGMS) in blood identifies differential DNA methylation in persons with late-onset dementia due to Alzheimer's disease (AD) but has not been tested in persons with mild cognitive impairment (MCI). METHODS: We used WGMS to compare DNA methylation levels at 25,244,219 CpG loci in 382 blood samples from 99 persons with MCI, 109 with AD, and 174 who are cognitively unimpaired (CU). RESULTS: WGMS identified 9,756 differentially methylated positions (DMPs) in persons with MCI, including 1,743 differentially methylated genes encoding proteins in biological pathways related to synapse organization, dendrite development, and ion transport. 447 DMPs exhibit progressively increasing or decreasing DNA methylation levels between CU, MCI, and AD that correspond to cognitive status. DISCUSSION: WGMS identifies DMPs in known and newly detected genes in blood from persons with MCI and AD that support blood DNA methylation levels as candidate biomarkers of cognitive status.

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.