Amyloid plaques and neurofibrillary tangles are the two main pathological hallmarks of Alzheimer disease (AD) [1]. While amyloid and tau deposition begins a decade or more prior to the first signs of memory loss, it is only after significant neuronal and synaptic loss has occurred in vulnerable brain regions that the first signs of cognitive impairment appear [2]. Pathological studies of AD and proposed models of disease progression suggest that synaptic loss is the best surrogate for cognitive decline in AD [3,4] as it appears to be more closely associated with cognitive outcomes than the degree of amyloid plaques, neurofibrillary tangles, or gliosis in AD brains [5].

Neurogranin (Ng) and synaptosome–associated protein-25 (SNAP-25) have recently been identified as potential cerebrospinal fluid (CSF) biomarkers of synaptic injury in AD [6,7]. Ng and SNAP-25 are synaptic proteins which are abundantly and preferentially expressed in the presynaptic (SNAP-25) or postsynaptic (Ng) membranes and are widely distributed in the human brain [8,9]. Ng is a neuron-specific [8] calmodulin-binding postsynaptic protein [10] which is abundantly expressed in neuronal dendritic spines. Several studies have implicated Ng in activity-dependent synaptic plasticity, memory, and learning [10-14]. Ng enhances synaptic function [8] and facilitates long-term potentiation by regulating the availability of calmodulin at synaptic sites [15-17]. SNAP-25 is a widely distributed presynaptic protein which is involved in docking and fusion of synaptic vesicles, a process essential for exocytosis [18]. SNAP-25 has also been implicated in axonal outgrowth and neurite elongation [19].

Studies by our group and others have shown that CSF Ng [7,20] and SNAP-25 [9,21] levels are elevated in AD compared to controls. Elevated CSF levels of synaptic proteins in AD likely reflect the release of abundant synaptic constituents into the extracellular space in the setting of neurodegeneration [7]. We have previously shown that CSF Ng levels strongly correlate with CSF levels of tau and tau phosphorylated at threonine 181 (p-tau181), whole brain and regional atrophy, and rates of cognitive decline in a large, well-characterized cohort of cognitively normal individuals and individuals with presymptomatic and early symptomatic AD. These individuals were enrolled in studies of aging and dementia at the Knight Washington University Alzheimer’s Disease Research Center and followed longitudinally for 2-3 years [7]. Furthermore, we have shown that CSF Ng levels correlate with cortical amyloid deposition in presymptomatic AD [7]. Importantly, in the previous cohort, CSF Ng offered a predictive value for future cognitive impairment in cognitively normal individuals over a 2-3 year follow-up period that was comparable to other biomarkers of AD pathology (CSF tau, p-tau181, and Aβ42). CSF Ng also complemented the collective ability of these markers to predict AD pathology in cognitively normal elderly individuals (ie, presymptomatic AD). Data from our group [22] and others [9,21], suggest that CSF SNAP-25 offers value as a diagnostic and predictive marker in early AD, and correlates with other CSF biomarkers of AD pathology. Together, these findings support the value of CSF Ng and SNAP-25 as CSF surrogates of synaptic injury in AD.

Functional magnetic resonance imaging (fMRI) studies have identified networks of cortical regions which demonstrate highly synchronized activity during the resting state or during the performance of specific cognitive tasks [23,24]. The default mode network (DMN), which includes the posterior cingulate, precuneus, medial temporal, medial prefrontal, and inferior parietal regions, is active during rest and shows reduced activity during cognitive tasks [25,26]. Reduced functional connectivity (FC) within the DMN has been shown in early AD, including mild cognitive impairment (MCI) due to AD [24,27,28].

Semantic memory refers to the recall of general facts and knowledge that are not contextually specific (eg, making a categorical or attributional judgment to a presented item) [29]. The neural correlates of semantic memory include a left lateralized network of cortical regions, including the posterior cingulate, precuneus, parahippocampal gyrus, posterior inferior parietal, middle temporal, fusiform, dorsomedial prefrontal, ventromedial prefrontal, and inferior frontal cortices [30,31]. Previous studies have shown the utility of the Famous Name Discrimination Task (FNDT) in evaluating FC within the semantic memory network in older adults, including those with early AD [32].

To our knowledge, no studies have investigated the utility of CSF Ng or SNAP-25 as surrogates of synaptic injury in functional imaging studies of healthy aging and AD. In this study, we propose to investigate associations between CSF Ng or SNAP-25 levels and FC measures in the default mode and semantic memory networks in cognitively normal older adults and those with early symptomatic AD, including MCI (Clinical Dementia Rating [CDR] 0.5) and mild dementia (CDR 1) due to AD. Findings from this study will support the utility of CSF Ng and SNAP-25 as fluid surrogates of synaptic injury in AD by evaluating their associations with functional imaging as an in vivo marker of synaptic integrity. CSF biomarkers that reflect functional alterations in neural networks targeted by early AD pathology (ie, default mode and semantic memory networks) will offer valuable tools to monitor disease progression and response to disease modifying therapies in clinical trials of AD therapeutics, independently of changes to amyloid or tau pathology, and will supplement information provided by cognitive and imaging outcome measures.

This will be a cross-sectional study of individuals with MCI due to AD or mild AD dementia (CDR 0.5-1; n=20), and cognitively normal controls (CDR 0; n=20). All participants will undergo a detailed clinical and neuropsychological assessment during the first visit, one lumbar puncture (LP) during the second visit, and one structural and functional MRI assessment during the third visit (Table 1). Functional MRI data will be acquired during resting state and performance of a semantic memory task (ie, FNDT). Cognitive, CSF, and MRI assessments will be completed within 4 months of enrollment. Resting state and task activated fMRI scans will be conducted in the same setting to minimize effects of environmental factors on fMRI parameters.

Participants will be recruited from the community and the Cognitive Neurology Clinic of the Ohio State University Wexner Medical Center. This study will include n=20 cognitively normal individuals (CDR 0), and n=20 individuals with a clinical diagnosis of single-domain or multi-domain amnestic MCI due to AD (CDR 0.5) or mild AD dementia (CDR 1). As some participants who enroll in the study may later elect to withdraw their participation (ie, drop out) or may be lost to follow-up, we anticipate the need to enroll a total of 50 participants (CDR 0, n=25; and CDR 0.5-1, n=25) to maintain adequate statistical power for the study.

The clinical diagnosis of amnestic MCI due to AD will be made according to standard clinical criteria as described by the National Institute on Aging and Alzheimer’s Association (NIA-AA) Working Group [33]. Clinical diagnoses will be supported by CSF biomarker data for tau, p-tau181, and Aβ42 (ie, the CSF biomarker phenotype of AD including elevated CSF tau or p-tau181, and low CSF Aβ42 levels) at the time of data analysis. This includes evaluation for other systemic or neurological disorders which could significantly contribute to cognitive impairment, and inclusion of results from ancillary structural imaging (brain computed tomography [CT] or structural MRI), neuropsychological testing, and ¹⁸F-fludeoxy-glucose positron emission tomography (FDG-PET) imaging (when available) into the diagnostic scheme [33].

The diagnosis of amnestic MCI will be based on impairment in episodic memory with or without impairment in other cognitive domains (ie, multi-domain and single-domain amnestic MCI, respectively), which is 1-1.5 SDs of age-, gender-, and education-matched norms, and is not associated with significant functional decline [33]. In most centers, a diagnosis of amnestic MCI due to AD is equivalent to a CDR of 0.5.

The clinical diagnosis of dementia due to AD will be made according to standard clinical criteria as described by the NIA-AA Working Group [34] and supported by CSF biomarker data for tau, p-tau181, and Aβ42. This will include evaluation for other disorders which could significantly contribute to cognitive impairment, and will also include results from ancillary structural imaging, neuropsychological testing, and FDG-PET imaging (when available) [34].

In individuals who meet standard criteria for dementia due to AD, the CDR will be used to determine the severity of dementia. A CDR designation of 1, 2, and 3 denotes mild, moderate, and severe AD dementia, respectively [35].

Normal cognition will be defined as cognitive performance on detailed neuropsychological assessments that falls within 1 SD of age-, gender-, and education-matched norms in all cognitive domains, and no subjective report of cognitive decline from an individual’s baseline (ie, CDR 0). CSF biomarker data will be used to differentiate cognitively normal controls who have no biomarker evidence of AD pathology (ie, those with normal CSF tau, p-tau181 and Aβ42 levels: CSF tau<350 pg/ml, p-tau181<50 pg/ml, and Aβ42>500 pg/ml) from cognitively normal controls who have biomarker evidence of AD pathology (ie, those with the CSF biomarker phenotype of AD: CSF tau≥350 pg/ml, p-tau181≥50 pg/ml, and Aβ42≤500 pg/ml) at the time of data analysis. These cutoff values are based on the CSF biomarker levels that provided the highest diagnostic accuracy (ie, combination of sensitivity and specificity as measured by the area under the curve [AUC] for the receiver operating characteristic [ROC] curves) in differentiating individuals with a clinical diagnosis of AD from cognitively normal controls in previous longitudinal studies of healthy aging and dementia at the Washington University’s Knight Alzheimer’s Disease Research Center [36].

Clinical assessments will be performed by neurologists and nurse practitioners in the Cognitive Neurology clinic of the Ohio State University. Clinical assessments will include a detailed review of the history of present illness, past medical, surgical, social, and family history, medications, allergies, and a detailed physical and neurological exam.

Neuropsychological assessments will be performed by experienced neuropsychometricians, and will include the following tests [37]: (1) Associate learning subtest of the Wechsler memory scale-IV [WMS-IV] [38]; (2) WMS-IV Logical Memory (I and II) [38]; (3) Hopkins Verbal Learning Test-Revised [39]; (4) Information subtest from the Wechsler adult intelligence scale-IV [WAIS-IV] [40]; (5) Boston naming test (short version) [41]; (6) animal fluency test [42]; (7) WMS-IV mental control (symbol span) [38]; (8) digit span forward and digit span backward (WAIS-IV) [40]; (9) letter fluency for F and S [43]; (10) block design (WAIS-IV) [40]; (11) digit symbol substitution tests [44]; (12) trail making tests A and B [45]; (13) the CDR [46]; (14) the Mini Mental Status Examination (MMSE) [47]; (15) the Self-Administered Gerocognitive Examination (SAGE) [48]; (16) the Geriatric Depression Scale (GDS) [49]; (17) the behavioral component of the Neuropsychiatric Inventory (NPI) [50]; and (18) the Functional Activity Questionnaire [51].

A total of 10 ml of blood will be obtained from each participant, collected in EDTA tubes, aliquoted, and frozen at –80°C. APOE genotyping will be performed using real-time PCR (polymerase chain reaction) on an Applied Biosystems 7900HT Real-Time PCR machine using the TaqMan SNP (single nucleotide polymorphism) Genotyping Assay (Applied Biosystems) for rs429358 and rs7412 as described [52].

Each participant will undergo one LP within 4 months of the clinical and neuropsychological assessments. A total of 20-25 ml will be obtained from each participant in the lateral decubitus position under sterile conditions, collected in sterile polypropylene tubes, centrifuged, aliquoted and placed on dry ice. CSF aliquots will be stored at –80°C, then thawed and centrifuged prior to analysis. CSF analyses of tau, p-tau181, and Aβ42 levels will be performed using the Innotest enzyme-linked immunoassay (Fujirebio, formerly Innogenetics) as described [7]. CSF analyses for Ng and SNAP-25 levels will be performed using a single molecule counting chemiluminescence assay (Erenna, Singulex) as described [7,53].

Student’s t tests, chi-square (X²) analyses, and analysis of covariance (ANCOVA) will examine differences in demographic, clinical, neuropsychological, CSF biomarker, and FC measures between the study groups (SPSSv15, SPSS, IL). Partial correlation analyses and linear regression models will examine associations between CSF biomarker levels and FC measures, adjusting for age, gender, and the APOE4 genotype (SPSSv15, SPSS, IL). Bootstrap analyses will compare correlations between CSF biomarker measures (individually or as combinations of markers, using principal components analysis) and FC in the DMN and semantic memory networks in AD and controls (R Statistical Software).

The main outcome measures of the study include CSF biomarker measurements (CSF Ng, SNAP-25, tau, p-tau181, and Aβ42 levels in pg/ml) and functional imaging measures including FC of the left and right precuneus seeds (ie, correlation between the timeseries of each of the left and right precuneus seeds and that of every voxel in the brain represented by voxel-wise partial correlation maps) during resting state and the performance of the semantic memory task (ie, FNDT). Analyses will be adjusted for covariates including age, gender, and the APOE4 genotype.