Microglial PLXDC2 Modulates Aβ Phagocytosis and Inflammatory Responses

S4-01-01: Identification of subgroups of Alzheimer disease

Alzheimer's disease (AD) lacks protein biomarkers reflective of its diverse underlying pathophysiology, hindering diagnostic and therapeutic advancements. Here, we used integrative proteomics to identify cerebrospinal fluid (CSF) biomarkers representing a wide spectrum of AD pathophysiology. Multiplex mass spectrometry identified ~3500 and ~12,000 proteins in AD CSF and brain, respectively. Network analysis of the brain proteome resolved 44 biologically diverse modules, 15 of which overlapped with the CSF proteome. CSF AD markers in these overlapping modules were collapsed into five protein panels representing distinct pathophysiological processes. Synaptic and metabolic panels were decreased in AD brain but increased in CSF, while glial-enriched myelination and immunity panels were increased in brain and CSF. The consistency and disease specificity of panel changes were confirmed in >500 additional CSF samples. These panels also identified biological subpopulations within asymptomatic AD. Overall, these results are a promising step toward a network-based biomarker tool for AD clinical applications.

Protein glycosylation is crucial for the central nervous system and brain functions, including processes that are defective in Alzheimer disease (AD) such as neurogenesis, synaptic function, and memory formation. Still, the roles of glycans in the development of AD are relatively unexplored. Glycomics studies of cerebrospinal fluid (CSF) have previously shown altered glycosylation pattern in patients with different stages of cognitive impairment, including AD, compared to healthy controls. As a consequence, we hypothesized that the glycan profile is altered in the brain of patients with AD and analyzed the asparagine‐linked (N‐linked) glycan profile in hippocampus and cortex in AD and control brain. Glycans were enzymatically liberated from brain glycoproteins and analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Eleven glycans showed significantly different levels in hippocampus compared to cortex in both control and AD brain. Two glycans in cortex and four in hippocampus showed different levels in AD compared to control brain. All glycans that differed between controls and AD brain had similar structures with one sialic acid, at least one fucose and a confirmed or potential bisecting N‐acetylglucosamine (GlcNAc). The glycans that were altered in AD brain differed from those that were altered in AD CSF. One glycan found to be present in significantly lower levels in both hippocampus and cortex in AD compared to control contained a structurally and functionally interesting epitope that we assign as a terminal galactose decorated with fucose and sialic acid. Altogether, these studies suggest that protein glycosylation is an important component in the development of AD and warrants further studies.

Although the pathophysiology of Alzheimer's disease (AD) and Parkinson's disease (PD) is unknown, altered brain antioxidative mechanisms have been found in both disorders. Ceruloplasmin (CP) and transferrin (TF) interact to limit concentrations of free ferrous iron (Fe2+), and thus play an important role in antioxidant defense in serum; both proteins are also produced in brain, where their significance as antioxidants is unknown. We quantified concentrations of CP and TF by immunoassay in AD (n = 17) and PD (n = 12) cerebrospinal fluid (CSF) to determine whether these proteins could serve as disease markers. CP was increased versus aged normal subjects (n = 11) in AD (p < 0.05) but not PD CSF, whereas TF concentrations did not differ between groups. CP levels have been reported to be elevated in some brain regions in AD, and increased CP in AD CSF may reflect this finding. Systemic inflammation and oxidative stress are major factors stimulating hepatic CP synthesis, and it remains to be determined whether increased CP concentrations in AD CSF and brain follow from similar mechanisms.

High Activities of BACE1 in Brains with Mild Cognitive Impairment

Alzheimer's disease (AD) is histopathologically characterized by the presence of numerous neurons with neurofibrillary tangles of paired helical filaments (PHF) in the neocortex, particularly the hippocampus. The major protein subunit of PHF, which also accumulate as neuropil threads and dystrophic neurites of the neuritic (senile) plaques, is the microtubule-associated protein tau. tau in AD brain is abnormally hyperphosphorylated. In addition to hyperphosphorylation, some of the AD tau is also ubiquitinated. The level of tau in AD brain is elevated severalfold, and this increase is in the form of the abnormally phospholylated tau. The levels of conjugated ubiquitin are also increased severalfold in AD brain. The cerebrospinal fluid (CSF) levels of both tau and ubiquitin are elevated in AD. However, some of the normal elderly and non-AD neurological control cases also show elevated levels of these two proteins in the CSF. At present, it is not clear whether the overlap in CSF tau or ubiquitin values between AD and the control groups is due to some unknown heterogeneity of AD, inclusion of presymptomatic AD cases in the control group, or lack of specificity of the CSF elevation of these markers to AD. Thus, until the results of prospective studies on many patients with AD and normal controls become available, the CSF tau and ubiquitin levels can be used only as additional tools in the psychometric and clinical diagnosis of AD.

Accumulation of Abeta protein in beta-amyloid deposits is a hallmark event in Alzheimer's disease (AD). Recent findings suggest anti-Abeta autoantibodies may have a role in AD pathology. However, a consensus has yet to emerge as to whether endogenous anti-Abeta autoantibodies are elevated, depressed, or unchanged in AD patients. Whereas experiments to date have used synthetic unmodified monomeric Abeta (Abetamon) to test autoimmunity, up to 40% of the Abeta pool inB AD brain consists of low molecular weight oligomeric cross-linked beta-amyloid protein species (CAPS). Recent studies also suggest that CAPS may be the primary neurotoxic agent in AD. In the present study, AD and nondemented control plasma were analyzed for immunoreactivity to CAPS and Abetamon. Plasma of both nondemented and AD patients were found to contain autoantibodies specific for soluble CAPS. Nondemented control and AD plasmas demonstrated similar immunoreactivity to Abetamon. In contrast, anti-CAPS antibodies in AD plasma were found to be significantly reduced compared with nondemented controls (p=0.018). Furthermore, age at onset for AD correlated significantly (p=0.041) with plasma immunoreactivity to CAPS. These data suggest that autoantibodies to CAPS are depleted in AD patients and raise the prospect that immunization with anti-CAPS antibodies might provide therapeutic benefit for AD.

Extracellular chaperones modulate the effects of Alzheimer’s patient cerebrospinal fluid on Aβ1-42 toxicity and uptake

Immunocytochemical staining was performed to investigate the presence of anti-hippocampal antibodies in cerebrospinal fluid (CSF) from patients with probable Alzheimer's disease (AD) (n = 19), aged normal controls (n = 9), and young normal controls (n = 10). Marked staining of neurons in the granule cell layer of the dentate gyrus and in pyramidal neurons in CA1-3 of the rat hippocampus was observed in 5 AD CSF samples (26%), 1 aged control sample (11%), and 1 young control sample (10%). These differences were not statistically significant. One of the immunoreactive AD CSF specimens also contained high concentrations of C5b-9, the membrane attack complex. The infrequent occurrence of anti-hippocampal antibodies in AD CSF, and the detection of similar immunoreactivity in control CSF specimens, suggest that these antibodies are unlikely to play a role in the neurodegenerative process in most individuals with AD. However, elevated C5b-9 concentration in an AD CSF specimen with marked immunoreactivity to hippocampal neurons suggests the possibility that anti-neuronal antibodies may contribute to complement activation in some AD patients.

Cerebrospinal fluid (CSF) tau is an excellent surrogate marker for assessing neuropathological changes that occur in Alzheimer's disease (AD) patients. However, whether the elevated tau in AD CSF is just a marker of neurodegeneration or, in fact, a part of the disease process is uncertain. Moreover, it is unknown how CSF tau relates to the recently described soluble high-molecular-weight (HMW) species that is found in the postmortem AD brain and can be taken up by neurons and seed aggregates. We have examined seeding and uptake properties of brain extracellular tau from various sources, including interstitial fluid (ISF) and CSF from an AD transgenic mouse model and postmortem ventricular and antemortem lumbar CSF from AD patients. We found that brain ISF and CSF tau from the AD mouse model can be taken up by cells and induce intracellular aggregates. Ventricular CSF from AD patients contained a rare HMW tau species that exerted a higher seeding activity. Notably, the HMW tau species was also detected in lumbar CSF from AD patients, and its levels were significantly elevated compared to control subjects. HMW tau derived from CSF of AD patients was seed competent in vitro. These findings suggest that CSF from an AD brain contains potentially bioactive HMW tau species, giving new insights into the role of CSF tau and biomarker development for AD. Ann Neurol 2016;80:355-367.

A disturbance of calcium homeostasis is believed to play an important role in the neurodegeneration of the brains of Alzheimer disease (AD) patients, but the molecular pathways by which it contributes to the disease are not well understood. Here we studied the activation of two major Ca(2+)-regulated brain proteins, calpain and calcineurin, in AD brain. We found that calpain I is activated, which in turn cleaves and activates calcineurin in AD brain. Mass spectrometric analysis indicated that the cleavage of calcineurin by calpain I is at lysine 501, a position C-terminal to the autoinhibitory domain, which produces a 57-kDa truncated form. The 57-kDa calcineurin maintains its Ca(2+)/calmodulin dependence of the phosphatase activity, but the phosphatase activity is remarkably activated upon truncation. The cleavage and activation of calcineurin correlate to the number of neurofibrillary tangles in human brains. These findings suggest that the overactivation of calpain I and calcineurin may mediate the role of calcium homeostatic disturbance in the neurodegeneration of AD.

Nitration of Tyrosine 10 Critically Enhances Amyloid β Aggregation and Plaque Formation

Neuronal pentraxin receptor in cerebrospinal fluid as a potential biomarker for neurodegenerative diseases

Oxidative stress, manifested by protein oxidation, lipid peroxidation, DNA oxidation and 3-nitrotyrosine formation, among other indices, is observed in Alzheimer's disease (AD) brain. Amyloid g -peptide (1-42) [A g (1-42)] may be central to the pathogenesis of AD. Our laboratory and others have implicated A g (1-42)-induced free radical oxidative stress in the neurodegeneration observed in AD brain. This paper reviews some of these studies from our laboratory. Recently, we showed both in-vitro and in-vivo that methionine residue 35 (Met-35) of A g (1-42) was critical to its oxidative stress and neurotoxic properties. Because the C-terminal region of A g (1-42) is helical, and invoking the i +4 rule of helices, we hypothesized that the carboxyl oxygen of lle-31, known to be within a van der Waals distance of the S atom of Met-35, would interact with the latter. This interaction could alter the susceptibility for oxidation of Met-35, i.e. free radical formation. Consistent with this hypothesis, substitution of lle-31 by the helix-breaking amino acid, proline, completely abrogated the oxidative stress and neurotoxic properties of A g (1-42). Removal of the Met-35 residue from the lipid bilayer by substitution of the negatively charged Asp for Gly-37 abrogated oxidative stress and neurotoxic properties of A g (1-42). The free radical scavenger vitamin E prevented A g (1-42)-induced ROS formation, protein oxidation, lipid peroxidation, and neurotoxicity in hippocampal neurons, consistent with our model for A g -associated free radical oxidative stress induced neurodegeneration in AD. ApoE, allele 4, is a risk factor for AD. Synaptosomes from apoE knock-out mice are more vulnerable to A g -induced oxidative stress (protein oxidation, lipid peroxidation, and ROS generation) than are those from wild-type mice. We also studied synaptosomes from allele-specific human apoE knock-in mice. Brain membranes from human apoE4 mice have greater vulnerability to A g (1-42)-induced oxidative stress than brain membranes from apoE2 or E3, assessed by the same indices, consistent with the notion of a coupling of the oxidative environment in AD brain and increased risk of developing this disorder. Using immunoprecipitation of proteins from AD and control brain obtained no longer than 4 h PMI, selective oxidized proteins were identified in the AD brain. Creatine kinase (CK) and g -actin have increased carbonyl groups, an index of protein oxidation, and Glt-1, the principal glutamate transporter, has increased binding of the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE). A g inhibits CK and causes lipid peroxidation, leading to HNE formation. Implications of these findings relate to decreased energy utilization, altered assembly of cytoskeletal proteins, and increased excitotoxicity to neurons by glutamate, all reported for AD. Other oxidatively modified proteins have been identified in AD brain by proteomics analysis, and these oxidatively-modified proteins may be related to increased excitotoxicity (glutamine synthetase), aberrant proteasomal degradation of damaged or aggregated proteins (ubiquitin C-terminal hydrolase L-1), altered energy production ( f -enolase), and diminished growth cone elongation and directionality (dihydropyrimindase-related protein 2). Taken together, these studies outlined above suggest that Met-35 is key to the oxidative stress and neurotoxic properties of A g (1-42) and may help explain the apoE allele dependence on risk for AD, some of the functional and structural alterations in AD brain, and strongly support a causative role of A g (1-42)-induced oxidative stress and neurodegeneration in AD.

BackgroundMild Cognitive Impairment (MCI) and Major Depressive Disorder (MDD) are independently associated with increased risk of dementia. Cerebrospinal fluid (CSF) and neuroimaging biomarkers can help to elucidate the etiology of cognitive impairment. We compared CSF biomarker profiles of Alzheimer’s disease (AD) and white matter hyperintensities (WMH) across three groups: MCI, MDD, or comorbid MCI+MDD.MethodWe measured CSF total tau, p‐tau, amyloid‐β42, using a sandwich ELISA method (Innotest, Fujirebio), and calculated p‐tau/amyloid‐β42 ratio in 31 participants diagnosed with MCI (N=13), MDD (N=7), or both MCI and MDD (N=11) enrolled in the Preventing Alzheimer’s dementia with cognitive remediation plus transcranial direct current stimulation in mild cognitive impairment and depression (PACt‐MD) study. All diagnoses were made in accordance with NIA‐AA and DSM 5 criteria. Participants with p‐tau &gt; 68 pg/mL and ATI &lt; 0.8 were considered AD (+). WMH were quantified on T2‐weighted magnetic resonance images in 27 of the participants. We compared CSF AD biomarkers across diagnostic groups. We then compared cognitive performance and WMH in those with AD (+) versus AD (‐) CSF biomarkers.Result9/31 participants exhibited AD (+) CSF: 7/13 with MCI and 2/11 with MCI+MDD. Participants with AD (+) CSF showed more impairment in verbal memory, working memory, language, and overall cognition than those with AD (‐) CSF (p=0.02, p=0.04, p=0.04, and p=0.03, respectively). 26/27 (96%) participants exhibited moderate to severe WMH irrespective of diagnosis or AD biomarker status.ConclusionFew participants in our sample with MDD had an AD (+) CSF biomarker profile, despite a neurocognitive profile of MCI. All participants with AD (‐) CSF had moderate to severe WMH, including those with MDD alone. Further investigation should determine whether volume or distribution of WMH contribute to cognitive impairment or depression in MCI patients who have an AD (‐) CSF biomarker profile.