Alzheimer’s disease (AD) is a devastating neurodegenerative disorder that stealthily begins its pathological development decades before the emergence of any clinical signs. This extended preclinical phase presents a crucial window of opportunity to biologically define and intervene in AD before irreversible brain damage occurs. It’s now understood that AD is not solely driven by amyloid and tau pathologies but is a multifactorial condition involving various pathways such as immunity, lipid metabolism, vascular dysfunction, and the endocytic pathway [1].
Cerebrospinal fluid (CSF), which bathes the brain and spinal cord, serves as a mirror reflecting the biochemical changes happening in the brain ante-mortem. Analyzing the CSF proteome can provide an in vivo pathobiological fingerprint of AD [2,3,4]. The CSF proteome is not static; protein levels fluctuate as AD progresses through its stages [2, 5]. A detailed examination of the CSF proteome in cognitively healthy individuals during the preclinical AD phase can unveil proteins and biological pathways particularly relevant to the origin and advancement of AD pathophysiology. These discoveries hold promise for identifying biomarkers to improve AD prognosis, pinpointing potential therapeutic targets, and establishing surrogate endpoints for clinical trials focusing on pre-dementia stages and diverse disease mechanisms [6, 7].
In a recent study, researchers analyzed over 900 CSF proteins to understand the mechanisms at play in the presymptomatic phase of AD. The study included 297 cognitively unimpaired individuals, categorized into amyloid negative (232) and amyloid positive (65), with a significant 72% undergoing clinical follow-up [8, 9]. Proximity extension assay (PEA) proteome data from 122 participants (103 amyloid positive and 19 amyloid negative) from the EMIF-AD preclinical cohort [10] was utilized to filter out proteins with inconsistent results and to validate the biomarker panel. The study aimed to: (i) define the biological changes characterizing preclinical AD; (ii) identify and validate a marker panel for preclinical AD and assess its predictive power for cognitive decline; and (iii) model marker levels against CSF Aβ42 levels to determine if biomarker changes precede or follow significant brain amyloidosis.
Figure 1: Study Design and Key Findings
Figure 1: Visual abstract outlining the study design and key findings. The CSF proteome analysis, focusing on proteins related to inflammation, proteolysis, and lipid transport, helps in defining preclinical AD and predicting its progression in cognitively unimpaired individuals.
The proteomic profiling identified 100 unique proteins that were differentially regulated in amyloid-positive compared to amyloid-negative cognitively unimpaired individuals. After excluding proteins with contradictory effects in the EMIF-AD dataset, the top 5 differentially regulated proteins were found to be involved in:
- Immune function: ITGB2, CCL11
- Protein glycosylation and folding: ENTPD5
- Insulin growth factor signaling pathway: IGFBP-1
- Protein phosphorylation: ABL1
Notably, CHIT1, a protein implicated in reactive gliosis and elevated in neurodegenerative dementias, showed the strongest correlation with amyloid pathology. This was followed by ITGB2, IGFBP-1, PRCP, and LGMN, with the latter two involved in lysosomal proteolytic function. These diverse proteins underscore the multifactorial nature of AD from its earliest stages. Interestingly, a significant majority (79 out of 100) of these dysregulated proteins in preclinical AD were also found to be dysregulated in symptomatic AD stages [2,3,4, 11, 12].
Functional enrichment analysis highlighted that these 100 CSF markers in preclinical AD are primarily linked to proteolysis and immune response, pathways known to be involved in AD pathophysiology and associated with amyloid and tau misfolding [1, 2]. This aligns with prior research indicating that immune function-related proteins are dysregulated years before the clinical onset of AD [5].
To create practical biomarker tools, the researchers condensed the proteomic data using statistical modeling, resulting in a 12-CSF protein panel. This panel demonstrated a high accuracy (AUC of 0.93) in detecting individuals in the preclinical phase of AD, which was further validated in the EMIF-AD cohort (AUC of 0.89). Consistent with the pathway enrichment analysis, most proteins in this panel are related to immune function (ITGB2, CXCL13, CLEC5A, CCL11, MCFD2, CRTAM, IL7). The panel also includes proteins associated with:
- Dopamine biosynthesis: DDC
- Lysosome activity: GLB1
- Protease inhibition: CST3 (Cystatin-C)
- Lipoprotein metabolism and lipid transport: IGFBP-1, PLTP
The inclusion of proteins like CLEC5A and ITGB2, which can regulate TYROBP/DAP12 – a key microglia network regulator in late-onset AD – highlights the potential role of these proteins in the earliest stages of AD. Many of these proteins, including ITGB2 and IGFBP-1, have also shown specificity for AD compared to other dementias [2]. The 12-CSF panel also effectively predicted progression to mild cognitive impairment (MCI) or dementia with 84% accuracy in clinically followed cases, increasing to 90% when predicting progression specifically to AD dementia. Panel positivity at baseline was linked to a significantly higher risk of clinical dementia (Hazard ratio = 8.37).
Modeling biomarker levels against CSF Aβ42 revealed that, except for GLB1 and MCFD2, all 12 panel proteins showed changes before CSF Aβ42 positivity, indicating their involvement in the earliest detectable stages of AD. This suggests that processes related to immune function, energy metabolism, neurotrophic function, and endolysosomal activity begin to shift even before significant amyloid buildup in the brain.
While these findings are promising, the study acknowledges limitations such as the limited number of cases progressing to AD dementia and the cross-sectional nature of the study. Future longitudinal studies and validation in larger independent cohorts are needed.
Conclusion: Defining a New Era in Early Alzheimer’s Detection
This CSF proteome profiling study provides compelling evidence that proteins involved in immune function, proteolysis, and lipoprotein metabolism are altered in the preclinical stages of Alzheimer’s disease. The identified 12-protein panel not only effectively defines preclinical AD with high accuracy but also predicts progression to AD dementia. The early changes in these proteins, some preceding amyloid detection in CSF, suggest they could be valuable targets for preventing amyloidosis and subsequent clinical symptoms. This panel, readily adaptable for custom assays, offers a powerful tool to monitor in vivo processes beyond amyloid and tau, paving the way for new therapeutic strategies and improved biomarker tools for clinical practice and trials aimed at combating Alzheimer’s disease in its earliest, most treatable phases.
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