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David M Holtzman

Publications and source records attributed to David M Holtzman.

At least 19 recordsLinked to original sources
PubMed2026

Priming of CD8+ T cells by peripheral dendritic cells exacerbates tau-mediated neurodegeneration.

Alzheimer's disease and primary tauopathies are marked by changes in adaptive immunity, with increased brain CD8+ T cells correlating with tau pathology severity. However, how peripheral T cells get primed to enter the brain and contribute to tau-mediated neurodegeneration remains unclear. In different disease conditions, conventional type 1 dendritic cells (cDC1s) cross-present antigens to prime CD8+ T cells into effector cells. We show that tauopathy mice lacking cDC1s or antigen cross-presentation are protected from neurodegeneration, with reduced brain CD8+ T cell infiltration and glial activation. The remaining CD8+ T cells exhibit limited clonal expansion, consistent with impaired priming. We further demonstrate that brain-derived antigens are presented in secondary lymphoid tissues, suggesting a site of T cell activation. Together, these findings establish cDC1-dependent peripheral priming as a key driver of CD8+ T cell accumulation in the brain and tau-mediated neurodegeneration.

Journal Article↗
PubMed2025

Lymphoid gene expression supports neuroprotective microglia function.

Microglia, the innate immune cells of the brain, play a defining role in the progression of Alzheimer's disease (AD)1. The microglial response to amyloid plaques in AD can range from neuroprotective to neurotoxic2. Here we show that the protective function of microglia is governed by the transcription factor PU.1, which becomes downregulated following microglial contact with plaques. Lowering PU.1 expression in microglia reduces the severity of amyloid disease pathology in mice and is linked to the expression of immunoregulatory lymphoid receptor proteins, particularly CD28, a surface receptor that is critical for T cell activation3,4. Microglia-specific deficiency in CD28, which is expressed by a small subset of plaque-associated PU.1low microglia, promotes a broad inflammatory microglial state that is associated with increased amyloid plaque load. Our findings indicate that PU.1low CD28-expressing microglia may operate as suppressive microglia that mitigate the progression of AD by reducing the severity of neuroinflammation. This role of CD28 and potentially other lymphoid co-stimulatory and co-inhibitory receptor proteins in governing microglial responses in AD points to possible immunotherapy approaches for treating the disease by promoting protective microglial functions.

Microglia↗
PubMed2025

Inhaled xenon modulates microglia and ameliorates disease in mouse models of amyloidosis and tauopathy.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. Antiamyloid antibody treatments modestly slow disease progression in mild dementia due to AD. Emerging evidence shows that homeostatic dysregulation of the brain immune system, especially that orchestrated by microglia, plays an important role in disease onset and progression. Thus, a major question is how to modulate the phenotype and function of microglia to treat AD. Xenon (Xe) gas is a noble gas used in human patients as an anesthetic and a neuroprotectant used for treating brain injuries. Xe penetrates the blood-brain barrier, which could make it an effective therapeutic. To assess the effect of Xe on microglia and AD pathology, we designed a custom Xe inhalation chamber and treated several mouse models of AD with Xe gas. Xe treatment induced mouse microglia to adopt an intermediate activation state that we have termed pre-neurodegenerative microglia (pre-MGnD). This microglial phenotypic transition was observed in mouse models of acute neurodegeneration and amyloidosis (APP/PS1 and 5xFAD mice) and tauopathy (P301S mice). This microglial state enhanced amyloid plaque compaction and reduced dystrophic neurites in the APP/PS1 and 5xFAD mouse models. Moreover, Xe inhalation reduced brain atrophy and neuroinflammation and improved nest-building behavior in P301S mice. Mechanistically, Xe inhalation induced homeostatic brain microglia toward a pre-MGnD state through IFN-γ signaling that maintained the microglial phagocytic response in APP/PS1 and 5xFAD mice while suppressing the microglial proinflammatory phenotype in P301S mice. These results support the translation of Xe inhalation as an approach for treating AD.

Animals↗
PubMed2012

Clinical and biomarker changes in dominantly inherited Alzheimer's disease.

BACKGROUND: The order and magnitude of pathologic processes in Alzheimer's disease are not well understood, partly because the disease develops over many years. Autosomal dominant Alzheimer's disease has a predictable age at onset and provides an opportunity to determine the sequence and magnitude of pathologic changes that culminate in symptomatic disease. METHODS: In this prospective, longitudinal study, we analyzed data from 128 participants who underwent baseline clinical and cognitive assessments, brain imaging, and cerebrospinal fluid (CSF) and blood tests. We used the participant's age at baseline assessment and the parent's age at the onset of symptoms of Alzheimer's disease to calculate the estimated years from expected symptom onset (age of the participant minus parent's age at symptom onset). We conducted cross-sectional analyses of baseline data in relation to estimated years from expected symptom onset in order to determine the relative order and magnitude of pathophysiological changes. RESULTS: Concentrations of amyloid-beta (Aβ)(42) in the CSF appeared to decline 25 years before expected symptom onset. Aβ deposition, as measured by positron-emission tomography with the use of Pittsburgh compound B, was detected 15 years before expected symptom onset. Increased concentrations of tau protein in the CSF and an increase in brain atrophy were detected 15 years before expected symptom onset. Cerebral hypometabolism and impaired episodic memory were observed 10 years before expected symptom onset. Global cognitive impairment, as measured by the Mini-Mental State Examination and the Clinical Dementia Rating scale, was detected 5 years before expected symptom onset, and patients met diagnostic criteria for dementia at an average of 3 years after expected symptom onset. CONCLUSIONS: We found that autosomal dominant Alzheimer's disease was associated with a series of pathophysiological changes over decades in CSF biochemical markers of Alzheimer's disease, brain amyloid deposition, and brain metabolism as well as progressive cognitive impairment. Our results require confirmation with the use of longitudinal data and may not apply to patients with sporadic Alzheimer's disease. (Funded by the National Institute on Aging and others; DIAN ClinicalTrials.gov number, NCT00869817.).

Age of Onset↗
PubMed2007

Cerebrospinal fluid tau/beta-amyloid(42) ratio as a prediction of cognitive decline in nondemented older adults.

OBJECTIVES: To investigate the ability of cerebrospinal fluid (CSF) and plasma measures to discriminate early-stage Alzheimer disease (AD) (defined by clinical criteria and presence/absence of brain amyloid) from nondemented aging and to assess whether these biomarkers can predict future dementia in cognitively normal individuals. DESIGN: Evaluation of CSF beta-amyloid(40) (Abeta(40)), Abeta(42), tau, phosphorylated tau(181), and plasma Abeta(40) and Abeta(42) and longitudinal clinical follow-up (from 1 to 8 years). SETTING: Longitudinal studies of healthy aging and dementia through an AD research center. PARTICIPANTS: Community-dwelling volunteers (n = 139) aged 60 to 91 years and clinically judged as cognitively normal (Clinical Dementia Rating [CDR], 0) or having very mild (CDR, 0.5) or mild (CDR, 1) AD dementia. RESULTS: Individuals with very mild or mild AD have reduced mean levels of CSF Abeta(42) and increased levels of CSF tau and phosphorylated tau(181). Cerebrospinal fluid Abeta(42) level completely corresponds with the presence or absence of brain amyloid (imaged with Pittsburgh Compound B) in demented and nondemented individuals. The CSF tau/Abeta(42) ratio (adjusted hazard ratio, 5.21; 95% confidence interval, 1.58-17.22) and phosphorylated tau(181)/Abeta(42) ratio (adjusted hazard ratio, 4.39; 95% confidence interval, 1.62-11.86) predict conversion from a CDR of 0 to a CDR greater than 0. CONCLUSIONS: The very mildest symptomatic stage of AD exhibits the same CSF biomarker phenotype as more advanced AD. In addition, levels of CSF Abeta(42), when combined with amyloid imaging, augment clinical methods for identifying in individuals with brain amyloid deposits whether dementia is present or not. Importantly, CSF tau/Abeta(42) ratios show strong promise as antecedent (preclinical) biomarkers that predict future dementia in cognitively normal older adults.

Aged↗
PubMed2006

Analysis of IFT74 as a candidate gene for chromosome 9p-linked ALS-FTD.

BACKGROUND: A new locus for amyotrophic lateral sclerosis--frontotemporal dementia (ALS-FTD) has recently been ascribed to chromosome 9p. METHODS: We identified chromosome 9p segregating haplotypes within two families with ALS-FTD (F476 and F2) and undertook mutational screening of candidate genes within this locus. RESULTS: Candidate gene sequencing at this locus revealed the presence of a disease segregating stop mutation (Q342X) in the intraflagellar transport 74 (IFT74) gene in family 476 (F476), but no mutation was detected within IFT74 in family 2 (F2). While neither family was sufficiently informative to definitively implicate or exclude IFT74 mutations as a cause of chromosome 9-linked ALS-FTD, the nature of the mutation observed within F476 (predicted to truncate the protein by 258 amino acids) led us to sequence the open reading frame of this gene in a large number of ALS and FTD cases (n = 420). An additional sequence variant (G58D) was found in a case of sporadic semantic dementia. I55L sequence variants were found in three other unrelated affected individuals, but this was also found in a single individual among 800 Human Diversity Gene Panel samples. CONCLUSION: Confirmation of the pathogenicity of IFT74 sequence variants will require screening of other chromosome 9p-linked families.

Amyotrophic Lateral Sclerosis↗
PubMed2006

Transport pathways for clearance of human Alzheimer's amyloid beta-peptide and apolipoproteins E and J in the mouse central nervous system.

Amyloid beta-peptide (Abeta) clearance from the central nervous system (CNS) maintains its low levels in brain. In Alzheimer's disease, Abeta accumulates in brain possibly because of its faulty CNS clearance and a deficient efflux across the blood-brain barrier (BBB). By using human-specific enzyme-linked immunosorbent assays, we measured a rapid 30 mins efflux at the BBB and transport via the interstitial fluid (ISF) bulk flow of human-unlabeled Abeta and of Abeta transport proteins, apolipoprotein E (apoE) and apoJ in mice. We show (i) Abeta40 is cleared rapidly across the BBB via low-density lipoprotein receptor-related protein (LRP)1 at a rate of 0.21 pmol/min g ISF or 6-fold faster than via the ISF flow; (ii) Abeta42 is removed across the BBB at a rate 1.9-fold slower compared with Abeta40; (iii) apoE, lipid-poor isoform 3, is cleared slowly via the ISF flow and across the BBB (0.03-0.04 pmol/min g ISF), and after lipidation its transport at the BBB becomes barely detectable within 30 mins; (iv) apoJ is eliminated rapidly across the BBB (0.16 pmol/min g ISF) via LRP2. Clearance rates of unlabeled and corresponding 125I-labeled Abeta and apolipoproteins were almost identical, but could not be measured at low physiologic levels by mass spectrometry. Amyloid beta-peptide 40 binding to apoE3 reduced its efflux rate at the BBB by 5.7-fold, whereas Abeta42 binding to apoJ enhanced Abeta42 BBB clearance rate by 83%. Thus, Abeta, apoE, and apoJ are cleared from brain by different transport pathways, and apoE and apoJ may critically modify Abeta clearance at the BBB.

Alzheimer Disease↗
PubMed2006

Matrix metalloproteinases expressed by astrocytes mediate extracellular amyloid-beta peptide catabolism.

It has been postulated that the development of amyloid plaques in Alzheimer's disease (AD) may result from an imbalance between the generation and clearance of the amyloid-beta peptide (Abeta). Although familial AD appears to be caused by Abeta overproduction, sporadic AD (the most prevalent form) may result from impairment in clearance. Recent evidence suggests that several proteases may contribute to the degradation of Abeta. Furthermore, astrocytes have recently been implicated as a potential cellular mediator of Abeta degradation. In this study, we examined the possibility that matrix metalloproteinases (MMPs), proteases known to be expressed and secreted by astrocytes, could play a role in extracellular Abeta degradation. We found that astrocytes surrounding amyloid plaques showed enhanced expression of MMP-2 and MMP-9 in aged amyloid precursor protein (APP)/presenilin 1 mice. Moreover, astrocyte-conditioned medium (ACM) degraded Abeta, lowering levels and producing several fragments after incubation with synthetic human Abeta(1-40) and Abeta(1-42). This activity was attenuated with specific inhibitors of MMP-2 and -9, as well as in ACM derived from mmp-2 or -9 knock-out (KO) mice. In vivo, significant increases in the steady-state levels of Abeta were found in the brains of mmp-2 and -9 KO mice compared with wild-type controls. Furthermore, pharmacological inhibition of the MMPs with N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001) increased brain interstitial fluid Abeta levels and elimination of half-life in APPsw mice. These results suggest that MMP-2 and -9 may contribute to extracellular brain Abeta clearance by promoting Abeta catabolism.

Amyloid beta-Peptides↗
PubMed2006

Apolipoprotein E and low density lipoprotein receptor-related protein facilitate intraneuronal Abeta42 accumulation in amyloid model mice.

The low density lipoprotein receptor-related protein (LRP) is highly expressed in the brain and has been shown to alter the metabolism of amyloid precursor protein and amyloid-beta peptide (Abeta) in vitro. Previously we developed mice that overexpress a functional LRP minireceptor (mLRP2) in their brains and crossed them to the PDAPP mouse model of Alzheimer disease. Overexpression of mLRP2 in 22-month-old PDAPP mice with amyloid plaques increased a pool of carbonate-soluble Abeta in the brain and worsened memory-related behavior. In the current study, we examined the effects of mLRP2 overexpression on 3-month-old PDAPP mice that had not yet developed amyloid plaques. We found significantly higher levels of membrane-associated Abeta42 in the hippocampus of mice that overexpressed mLRP2. Using immunohistochemical methods, we observed significant intraneuronal Abeta42 in the hippocampus and frontal cortex of PDAPP mice, which frequently co-localized with the lysosomal marker LAMP-1. Interestingly, PDAPP mice lacking apolipoprotein E (apoE) had much less intraneuronal Abeta42. We also found that PC12 cells overexpressing mLRP2 cleared Abeta42 and Abeta40 more rapidly from media than PC12 cells transfected with the vector only. Preincubation of apoE3 or apoE4 with Abeta42 increased the rate of Abeta clearance, and this effect was partially blocked by receptor-associated protein. Our results support the hypothesis that LRP binds and endocytoses Abeta42 both directly and via apoE but that endocytosed Abeta42 is not completely degraded and accumulates in intraneuronal lysosomes.

Amyloid beta-Peptides↗
PubMed2006

Pomegranate juice decreases amyloid load and improves behavior in a mouse model of Alzheimer's disease.

Although there are no proven ways to delay onset or slow progression of Alzheimer's disease (AD), studies suggest that diet can affect risk. Pomegranates contain very high levels of antioxidant polyphenolic substances as compared to other fruits and vegetables. Polyphenols have been shown to be neuroprotective in different model systems. We asked whether dietary supplementation with pomegranate juice (PJ) would influence behavior and AD-like pathology in a transgenic mouse model. Transgenic mice (APP(sw)/Tg2576) received either PJ or sugar water control from 6 to 12.5 months of age. PJ-treated mice learned water maze tasks more quickly and swam faster than controls. Mice treated with PJ had significantly less (approximately 50%) accumulation of soluble Abeta42 and amyloid deposition in the hippocampus as compared to control mice. These results suggest that further studies to validate and determine the mechanism of these effects, as well as whether substances in PJ may be useful in AD, should be considered.

Alzheimer Disease↗
PubMed2006

Role of the Menkes copper-transporting ATPase in NMDA receptor-mediated neuronal toxicity.

Menkes disease, a fatal neurodegenerative disorder resulting in seizures, hypotonia, and failure to thrive, is due to inherited loss-of-function mutations in the gene encoding a copper-transporting ATPase (Atp7a) on the X chromosome. Although affected patients exhibit signs and symptoms of copper deficiency, the mechanisms resulting in neurologic disease remain unknown. We recently discovered that Atp7a is required for the production of an NMDA receptor-dependent releasable copper pool within hippocampal neurons, a finding that suggests a role for copper in activity-dependent modulation of synaptic activity. In support of this hypothesis, we now demonstrate that copper chelation exacerbates NMDA-mediated excitotoxic cell death in primary hippocampal neurons, whereas the addition of copper is specifically protective and results in a significant decrease in cytoplasmic Ca(2+) levels after NMDA receptor activation. Consistent with the known neuroprotective effect of NMDA receptor nitrosylation, we show here that this protective effect of copper depends on endogenous nitric oxide production in hippocampal neurons, demonstrating in vivo links among neuroprotection, copper metabolism, and nitrosylation. Atp7a is required for these copper-dependent effects: Hippocampal neurons isolated from newborn Mo(br) mice reveal a marked sensitivity to endogenous glutamate-mediated NMDA receptor-dependent excitotoxicity in vitro, and mild hypoxic/ischemic insult to these mice in vivo results in significantly increased caspase 3 activation and neuronal injury. Taken together, these data reveal a unique connection between copper homeostasis and NMDA receptor activity that is of broad relevance to the processes of synaptic plasticity and excitotoxic cell death.

Adenosine Triphosphatases↗
PubMed2006

National Institute of Neurological Disorders and Stroke-Canadian Stroke Network vascular cognitive impairment harmonization standards.

BACKGROUND AND PURPOSE: One in 3 individuals will experience a stroke, dementia or both. Moreover, twice as many individuals will have cognitive impairment short of dementia as either stroke or dementia. The commonly used stroke scales do not measure cognition, while dementia criteria focus on the late stages of cognitive impairment, and are heavily biased toward the diagnosis of Alzheimer disease. No commonly agreed standards exist for identifying and describing individuals with cognitive impairment, particularly in the early stages, and especially with cognitive impairment related to vascular factors, or vascular cognitive impairment. METHODS: The National Institute for Neurological Disorders and Stroke (NINDS) and the Canadian Stroke Network (CSN) convened researchers in clinical diagnosis, epidemiology, neuropsychology, brain imaging, neuropathology, experimental models, biomarkers, genetics, and clinical trials to recommend minimum, common, clinical and research standards for the description and study of vascular cognitive impairment. RESULTS: The results of these discussions are reported herein. CONCLUSIONS: The development of common standards represents a first step in a process of use, validation and refinement. Using the same standards will help identify individuals in the early stages of cognitive impairment, will make studies comparable, and by integrating knowledge, will accelerate the pace of progress.

Brain↗
PubMed2006

Human amyloid-beta synthesis and clearance rates as measured in cerebrospinal fluid in vivo.

Certain disease states are characterized by disturbances in production, accumulation or clearance of protein. In Alzheimer disease, accumulation of amyloid-beta (Abeta) in the brain and disease-causing mutations in amyloid precursor protein or in enzymes that produce Abeta indicate dysregulation of production or clearance of Abeta. Whether dysregulation of Abeta synthesis or clearance causes the most common form of Alzheimer disease (sporadic, >99% of cases), however, is not known. Here, we describe a method to determine the production and clearance rates of proteins within the human central nervous system (CNS). We report the first measurements of the fractional production and clearance rates of Abeta in vivo in the human CNS to be 7.6% per hour and 8.3% per hour, respectively. This method may be used to search for novel biomarkers of disease, to assess underlying differences in protein metabolism that contribute to disease and to evaluate treatments in terms of their pharmacodynamic effects on proposed disease-causing pathways.

Amino Acid Sequence↗
PubMed2006

Matrix metalloproteinase-9 degrades amyloid-beta fibrils in vitro and compact plaques in situ.

The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-beta fibrils (fAbeta), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble Abeta (sAbeta) could degrade fAbeta as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fAbeta and that this ability is not shared by other sAbeta-degrading enzymes examined, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin. fAbeta was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fAbeta breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fAbeta were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of Abeta that are cleaved during its degradation. Only MMP-9 digests contained fragments (Abeta(1-20) and Abeta(1-30)) from fAbeta(1-42) substrate; the corresponding cleavage sites are thought to be important for beta-pleated sheet formation. To determine whether MMP-9 can degrade plaques formed in vivo, fresh brain slices from aged APP/PS1 mice were incubated with proteases. MMP-9 digestion resulted in a decrease in thioflavin-S (ThS) staining. Consistent with a role for endogenous MMP-9 in this process in vivo, MMP-9 immunoreactivity was detected in astrocytes surrounding amyloid plaques in the brains of aged APP/PS1 and APPsw mice, and increased MMP activity was selectively observed in compact ThS-positive plaques. These findings suggest that MMP-9 can degrade fAbeta and may contribute to ongoing clearance of plaques from amyloid-laden brains.

Aging↗
PubMed2006

Caspase-3 deficiency during development increases vulnerability to hypoxic-ischemic injury through caspase-3-independent pathways.

Neonatal hypoxia-ischemia (H-I) is a common cause of perinatal morbidity and mortality leading to prominent activation of caspase-3 in the brain. Previous studies have shown that acute inhibition of caspase-3 can protect against neonatal H-I in rats. In this study, we investigated brain injury following neonatal H-I in mice deficient in caspase-3. Wild-type, caspase-3+/- and caspase-3-/- mice underwent unilateral carotid ligation at postnatal day (P) 7, followed by 45 min of exposure to 8% oxygen. Surprisingly, tissue loss at P14 was significantly higher in caspase-3-/- mice when compared to wild-type littermates. As in rats, we found that acute inhibition of caspase-3 in mice leads to decrease in tissue loss at P14. There was no difference in nuclear morphology, DNA laddering or calpain activation between caspase-3-/-caspase-3+/- and wild-type mice subjected to H-I, and there was no evidence for compensatory activation of other caspases in caspase-3-/- mice. Also, all genotypes showed evidence of mitochondrial dysfunction after H-I, suggesting that this is a critical point in regulation of neuronal cell death following neonatal H-I. Our results suggest that long-term inhibition of caspase-3 during development, unlike acute inhibition, leads to upregulation of caspase-3-independent cell death pathways and increases the vulnerability of the developing brain to neonatal H-I injury.

Animals↗
PubMed2006

Inverse relation between in vivo amyloid imaging load and cerebrospinal fluid Abeta42 in humans.

OBJECTIVES: Amyloid-beta(42) (Abeta(42)) appears central to Alzheimer's disease (AD) pathogenesis and is a major component of amyloid plaques. Mean cerebrospinal fluid (CSF) Abeta(42) is decreased in dementia of the Alzheimer's type. This decrease may reflect plaques acting as an Abeta(42) "sink," hindering transport of soluble Abeta(42) between brain and CSF. We investigated this hypothesis. METHODS: We compared the in vivo brain amyloid load (via positron emission tomography imaging of the amyloid-binding agent, Pittsburgh Compound-B [PIB]) with CSF Abeta(42) and other measures (via enzyme-linked immunosorbent assay) in clinically characterized research subjects. RESULTS: Subjects fell into two nonoverlapping groups: those with positive PIB binding had the lowest CSF Abeta(42) level, and those with negative PIB binding had the highest CSF Abeta(42) level. No relation was observed between PIB binding and CSF Abeta(40), tau, phospho-tau(181), plasma Abeta(40), or plasma Abeta(42). Importantly, PIB binding and CSF Abeta(42) did not consistently correspond with clinical diagnosis; three cognitively normal subjects were PIB-positive with low CSF Abeta(42), suggesting the presence of amyloid in the absence of cognitive impairment (ie, preclinical AD). INTERPRETATION: These observations suggest that brain amyloid deposition results in low CSF Abeta(42), and that amyloid imaging and CSF Abeta(42) may potentially serve as antecedent biomarkers of (preclinical) AD.

Aged↗
PubMed2006

Quantitative analysis of amyloid beta peptides in cerebrospinal fluid of Alzheimer's disease patients by immunoaffinity purification and stable isotope dilution liquid chromatography/negative electrospray ionization tandem mass spectrometry.

The 40 and 42 amino-acid residue forms of amyloid beta (Abeta(1-40) and Abeta(1-42)) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Abeta peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Abeta peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [15N]-labeled Abeta peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/MS system. The validated method had limits of quantitation of 44 fmol/mL (200 pg/mL) for Abeta(1-42) and 92 fmol/mL (400 pg/mL) for Abeta(1-40). An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Abeta(1-42) in human CSF (r2 = 0.915), although less correlation was observed for Abeta(1-40) (r2 = 0.644). Mean CSF Abeta(1-42) concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 +/- 0.25 ng/mL; n = 7). Abeta(1-40) concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 +/- 3.07 ng/mL; n = 7). Consistent with literature reports, mean Abeta(1-42) concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 +/- 0.59 ng/mL; n = 7), whereas there was no difference in Abeta(1-40) concentrations between AD patients and normal subjects (mean 5.88 +/- 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Abeta(1-42) concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.

Alzheimer Disease↗