Madhav Thambisetty

Association of Plasma Clusterin Concentration With Severity, Pathology, and Progression in Alzheimer Disease

CONTEXT Blood-based analytes may be indicators of pathological processes in Alzheimer disease (AD). OBJECTIVE To identify plasma proteins associated with AD pathology using a combined proteomic and neuroimaging approach. DESIGN Discovery-phase proteomics to identify plasma proteins associated with correlates of AD pathology. Confirmation and validation using immunodetection in a replication set and an animal model. SETTING A multicenter European study (AddNeuroMed) and the Baltimore Longitudinal Study of Aging. PARTICIPANTS Patients with AD, subjects with mild cognitive impairment, and healthy controls with standardized clinical assessments and structural neuroimaging. MAIN OUTCOME MEASURES Association of plasma proteins with brain atrophy, disease severity, and rate of clinical progression. Extension studies in humans and transgenic mice tested the association between plasma proteins and brain amyloid. RESULTS Clusterin/apolipoprotein J was associated with atrophy of the entorhinal cortex, baseline disease severity, and rapid clinical progression in AD. Increased plasma concentration of clusterin was predictive of greater fibrillar amyloid-beta burden in the medial temporal lobe. Subjects with AD had increased clusterin messenger RNA in blood, but there was no effect of single-nucleotide polymorphisms in the gene encoding clusterin with gene or protein expression. APP/PS1 transgenic mice showed increased plasma clusterin, age-dependent increase in brain clusterin, as well as amyloid and clusterin colocalization in plaques. CONCLUSIONS These results demonstrate an important role of clusterin in the pathogenesis of AD and suggest that alterations in amyloid chaperone proteins may be a biologically relevant peripheral signature of AD.

Longitudinal Changes in Cortical Thickness Associated with Normal Aging

Imaging studies of anatomic changes in regional gray matter volumes and cortical thickness have documented age effects in many brain regions, but the majority of such studies have been cross-sectional investigations of individuals studied at a single point in time. In this study, using serial imaging assessments of participants in the Baltimore Longitudinal Study of Aging (BLSA), we investigate longitudinal changes in cortical thickness during aging in a cohort of 66 older adults (mean age 68.78; sd. 6.6; range 60-84 at baseline) without dementia. We used the Cortical Reconstruction Using Implicit Surface Evolution CRUISE suite of algorithms to automatically generate a reconstruction of the cortical surface and identified twenty gyral based regions of interest per hemisphere. Using mixed effects regression, we investigated longitudinal changes in these regions over a mean follow-up interval of 8 years. The main finding in this study is that age-related decline in cortical thickness is widespread, but shows an anterior-posterior gradient with frontal and parietal regions, in general, exhibiting greater rates of decline than temporal and occipital. There were fewer regions in the right hemisphere showing statistically significant age-associated longitudinal decreases in mean cortical thickness. Males showed greater rates of decline in the middle frontal, inferior parietal, parahippocampal, postcentral, and superior temporal gyri in the left hemisphere, right precuneus and bilaterally in the superior parietal and cingulate regions. Significant nonlinear changes over time were observed in the postcentral, precentral, and orbitofrontal gyri on the left and inferior parietal, cingulate, and orbitofrontal gyri on the right.

Fulminant idiopathic intracranial hypertension

Objective: To describe the incidence and characteristics of acute and rapidly progressive visual loss in idiopathic intracranial hypertension (IIH). Methods: We reviewed the medical records of all patients with IIH seen at two institutions. “Fulminant IIH” was defined as the acute onset of symptoms and signs of intracranial hypertension (less than 4 weeks between onset of initial symptoms and severe visual loss), rapid worsening of visual loss over a few days, and normal brain MRI and MR venography (or CT venogram). Results: Sixteen cases with “fulminant IIH” were included (16 women, mean age 23.8 years [range 14 to 39 years]). All were obese. One patient had iron-deficiency anemia, four had systemic hypertension, and none had known sleep apnea syndrome. Acute or subacute headache, nausea and vomiting, and visual loss were present in all patients. The first lumbar puncture performed for the diagnosis showed a mean CSF opening pressure of 54.1 cm H2O (range 29 to 60 cm H2O). In addition to the initial lumbar puncture, medical treatment included acetazolamide (1 to 2 g/day) in all patients, and IV methylprednisolone in four patients. Repeat lumbar punctures were performed in 11 of the 16 patients. Surgical treatment (optic nerve sheath fenestration in five cases, lumboperitoneal CSF shunting procedure in nine cases, and ventriculoperitoneal shunting procedure in two cases) was performed because of ongoing visual loss in all cases. The median delay between evaluation in neuro-ophthalmology and surgery was 3 days (range a few hours to 37 days). All patients reported dramatic improvement of headaches and vomiting following surgery. Visual function improved in 14 cases, although 8 patients (50%) remained legally blind. Visual fields remained severely altered in all cases. Conclusion: Severe and rapidly progressive visual loss suggests “fulminant idiopathic intracranial hypertension” and should prompt aggressive management. Urgent surgery may be required in these patients, and temporizing measures such as repeat lumbar punctures, lumbar drainage, and IV steroids considered.

Plasma clusterin concentration is associated with longitudinal brain atrophy in mild cognitive impairment

Recent genetic and proteomic studies demonstrate that clusterin/apolipoprotein-J is associated with risk, pathology, and progression of Alzheimers disease (AD). Our main aim was to examine associations between plasma clusterin concentration and longitudinal changes in brain volume in normal aging and mild cognitive impairment (MCI). A secondary objective was to examine associations between peripheral concentration of clusterin and its concentration in the brain within regions that undergo neuropathological changes in AD. Non-demented individuals (N=139; mean baseline age 70.5 years) received annual volumetric MRI (912 MRI scans in total) over a mean six-year interval. Sixteen participants (92 MRI scans in total) were diagnosed during the course of the study with amnestic MCI. Clusterin concentration was assayed by ELISA in plasma samples collected within a year of the baseline MRI. Mixed effects regression models investigated whether plasma clusterin concentration was associated with rates of brain atrophy for control and MCI groups and whether these associations differed between groups. In a separate autopsy sample of individuals with AD (N=17) and healthy controls (N=4), we examined the association between antemortem clusterin concentration in plasma and postmortem levels in the superior temporal gyrus, hippocampus and cerebellum. The associations of plasma clusterin concentration with rates of change in brain volume were significantly different between MCI and control groups in several volumes including whole brain, ventricular CSF, temporal gray matter as well as parahippocampal, superior temporal and cingulate gyri. Within the MCI but not control group, higher baseline concentration of plasma clusterin was associated with slower rates of brain atrophy in these regions. In the combined autopsy sample of AD and control cases, representing a range of severity in AD pathology, we observed a significant association between clusterin concentration in the plasma and that in the superior temporal gyrus. Our findings suggest that clusterin, a plasma protein with roles in amyloid clearance, complement inhibition and apoptosis, is associated with rate of brain atrophy in MCI. Furthermore, peripheral concentration of clusterin also appears to reflect its concentration within brain regions vulnerable to AD pathology. These findings in combination suggest an influence of this multi-functional protein on early stages of progression in AD pathology.

Evidence of altered phosphatidylcholine metabolism in Alzheimer's disease

Abberant lipid metabolism is implicated in Alzheimers disease (AD) pathophysiology, but the connections between AD and lipid metabolic pathways are not fully understood. To investigate plasma lipids in AD, a multiplatform screen (n = 35 by liquid chromatography-mass spectrometry and n = 35 by nuclear magnetic resonance) was developed, which enabled the comprehensive analysis of plasma from 3 groups (individuals with AD, individuals with mild cognitive impairment (MCI), and age-matched controls). This screen identified 3 phosphatidylcholine (PC) molecules that were significantly diminished in AD cases. In a subsequent validation study (n = 141), PC variation in a bigger sample set was investigated, and the same 3 PCs were found to be significantly lower in AD patients: PC 16:0/20:5 (p < 0.001), 16:0/22:6 (p < 0.05), and 18:0/22:6 (p < 0.01). A receiver operating characteristic (ROC) analysis of the PCs, combined with apolipoprotein E (ApoE) data, produced an area under the curve predictive value of 0.828. Confirmatory investigations into the background biochemistry indiciated no significant change in plasma levels of 3 additional PCs of similar structure, total choline containing compounds or total plasma omega fatty acids, adding to the evidence that specific PCs play a role in AD pathology.

APOE ε4 Genotype and Longitudinal Changes in Cerebral Blood Flow in Normal Aging

OBJECTIVE To study differences in longitudinal changes in regional cerebral blood flow (rCBF) between apolipoprotein E (APOE) epsilon4 carriers and noncarriers in nondemented older adults from the Baltimore Longitudinal Study of Aging using positron emission tomography in order to determine whether there are regionally specific longitudinal changes in rCBF in APOE epsilon4 carriers that might be related to its well-established role as a genetic risk factor for Alzheimer disease. DESIGN, SETTING, AND PARTICIPANTS Using oxygen 15 ([(15)O])-labeled water positron emission tomography and voxel-based analysis, we compared changes in rCBF over an 8-year period between 29 nondemented APOE epsilon4 carriers and 65 noncarriers older than 55 years. Serial neuropsychological data were collected for all participants. RESULTS Widespread differences were observed in longitudinal change in rCBF between epsilon4 carriers and noncarriers. The predominant pattern was greater rCBF decline in epsilon4 carriers. These differences were observed in the frontal, parietal, and temporal cortices. The affected brain regions were those especially vulnerable to pathological changes in Alzheimer disease. Both epsilon4 carriers and noncarriers remained free of clinical diagnoses of dementia or mild cognitive impairment during the course of the study. CONCLUSIONS Our findings suggest that APOE epsilon4-mediated risk for Alzheimer disease is associated with widespread decline in rCBF over time that precedes the onset of dementia. Accelerated rates of decline in brain function in APOE epsilon4 carriers may contribute to an increased risk for Alzheimer disease and a younger age at onset.

Plasma Biomarkers of Brain Atrophy in Alzheimer's Disease

Peripheral biomarkers of Alzheimers disease (AD) reflecting early neuropathological change are critical to the development of treatments for this condition. The most widely used indicator of AD pathology in life at present is neuroimaging evidence of brain atrophy. We therefore performed a proteomic analysis of plasma to derive biomarkers associated with brain atrophy in AD. Using gel based proteomics we previously identified seven plasma proteins that were significantly associated with hippocampal volume in a combined cohort of subjects with AD (N = 27) and MCI (N = 17). In the current report, we validated this finding in a large independent cohort of AD (N = 79), MCI (N = 88) and control (N = 95) subjects using alternative complementary methods—quantitative immunoassays for protein concentrations and estimation of pathology by whole brain volume. We confirmed that plasma concentrations of five proteins, together with age and sex, explained more than 35% of variance in whole brain volume in AD patients. These proteins are complement components C3 and C3a, complement factor-I, γ-fibrinogen and alpha-1-microglobulin. Our findings suggest that these plasma proteins are strong predictors of in vivo AD pathology. Moreover, these proteins are involved in complement activation and coagulation, providing further evidence for an intrinsic role of these pathways in AD pathogenesis.

Proteome-based identification of plasma proteins associated with hippocampal metabolism in early Alzheimer’s disease

Background and methodsThere is an urgent need for peripheral surrogates of Alzheimer’s disease (AD) that accurately reflect disease state and severity as well as correlate with key features of its neuropathology. The aim of this study was to identify plasma proteins associated with known in vivo markers of disease activity. In an earlier proteomic study of plasma, we discovered a panel of 15 proteins that were differentially expressed in AD and further validated complement factor-H (CFH) and alpha-2-macroglobulin (A2M) as AD-specific plasma biomarkers. In the present study, we extended these findings by testing the associations of these plasma proteins with neuro-imaging measures of disease progression in AD. We combined 1H-magnetic resonance spectroscopy of the hippocampus and MRI-based hippocampal volumetry with proteomic analysis of plasma in early AD and mild cognitive impairment (MCI) to achieve this goal. Using 1H-magnetic resonance spectroscopy, we derived estimates of the hippocampal metabolite ratio N-acetylaspartate/myo-inositol (NAA/mI), a biochemical measure that is associated with cognitive decline in early AD. We also undertook a proteomic analysis of plasma in these individuals using two-dimensional gel electrophoresis (2DGE).ResultsWe observed that two plasma proteins previously shown to be differentially expressed in AD, complement factor-H (CFH) and alpha-2-macroglobulin (A2M) showed significant positive correlations with hippocampal NAA/mI ratio in AD.ConclusionsThe association of plasma CFH and A2M with hippocampal NAA/mI in this cohort of AD subjects suggests that these proteins may reflect disease progression in early AD. These findings warrant validation in large population-based datasets.

Proteome-based plasma markers of brain amyloid-β deposition in non-demented older individuals.

Blood-based markers reflecting core pathological features of Alzheimers disease (AD) in pre-symptomatic individuals are likely to accelerate the development of disease-modifying treatments. Our aim was to discover plasma proteins associated with brain amyloid-β (Aβ) burden in non-demented older individuals. We performed discovery-phase experiments using two dimensional gel electrophoresis (2DGE) and mass spectrometry-based proteomic analysis of plasma in combination with 11C-PiB PET imaging of the brain in samples collected 10 years prior to the PET scans. Confirmatory studies used ELISA assays in a separate set of blood samples obtained within a year of the PET scans. We observed that a panel of 18 2DGE plasma protein spots effectively discriminated between individuals with high and low brain Aβ. Mass spectrometry identified these proteins, many of which have established roles in Aβ clearance, including a strong signal from apolipoprotein-E (ApoE). In validation-phase studies, we observed a strong association between plasma ApoE concentration and Aβ burden in the medial temporal lobe. Targeted voxel-based analysis localized this association to the hippocampus and entorhinal cortex. APOE ε4 carriers also showed greater Aβ levels in several brain regions relative to ε4 non-carriers. These results suggest that both peripheral concentration of ApoE protein and APOE genotype are related to early neuropathological changes in brain regions vulnerable to AD pathology even in the non-demented elderly. Our strategy combining proteomics with in vivo brain amyloid imaging holds promise for the discovery of biologically relevant peripheral markers in those at risk for AD.

Pharmacologic inhibition of ROCK2 suppresses amyloid-β production in an Alzheimer's disease mouse model.

Alzheimers disease (AD) is the leading cause of dementia and has no cure. Genetic, cell biological, and biochemical studies suggest that reducing amyloid-β (Aβ) production may serve as a rational therapeutic avenue to delay or prevent AD progression. Inhibition of RhoA, a Rho GTPase family member, is proposed to curb Aβ production. However, a barrier to this hypothesis has been the limited understanding of how the principal downstream effectors of RhoA, Rho-associated, coiled-coil containing protein kinase (ROCK) 1 and ROCK2, modulate Aβ generation. Here, we report that ROCK1 knockdown increased endogenous human Aβ production, whereas ROCK2 knockdown decreased Aβ levels. Inhibition of ROCK2 kinase activity, using an isoform-selective small molecule (SR3677), suppressed β-site APP cleaving enzyme 1 (BACE1) enzymatic action and diminished production of Aβ in AD mouse brain. Immunofluorescence and confocal microscopy analyses revealed that SR3677 alters BACE1 endocytic distribution and promotes amyloid precursor protein (APP) traffic to lysosomes. Moreover, SR3677 blocked ROCK2 phosphorylation of APP at threonine 654 (T654); in neurons, T654 was critical for APP processing to Aβ. These observations suggest that ROCK2 inhibition reduces Aβ levels through independent mechanisms. Finally, ROCK2 protein levels were increased in asymptomatic AD, mild cognitive impairment, and AD brains, demonstrating that ROCK2 levels change in the earliest stages of AD and remain elevated throughout disease progression. Collectively, these findings highlight ROCK2 as a mechanism-based therapeutic target to combat Aβ production in AD.