It is ascertained that synaptic dysfunction represents an early pathological event that precede neurodegeneration in Alzheimer’ s disease (AD), even though the molecular mechanisms that underlie synapse dysfunction remain to be completely understood [1,2]. Nonetheless, biomarkers able to detect changes in synaptic function in vivo are highly relevant as they have the potential to reveal early stages’ changes in patients and to track the target engagement of specific disease-modifying strategies. A range of CSF synapse-related biomarkers including neurogranin, GAP43, SNAP25, neuregulin-1, PSD-95 and neuronal pentraxin have indeed been reported in AD at variance with other dementing illnesses [1-3].
To identify reliable and reproducible synaptic biomarker for AD, we focused on the cyclase-associated protein 2 (CAP2), a postsynaptic actin-binding protein that governs actin cytoskeleton dynamics in dendritic spines. CAP2 is expressed in the brain and in a limited range of tissues. In particular, CAP2 downregulation differently affects neuronal and dendritic spine morphology in cerebral cortex and hippocampus, suggesting a brain area-specificity [4-5]. In excitatory neurons, CAP2 regulates cofilin activity and actin cytoskeleton dynamics in spines. CAP2 dimerization is is crucial for the long-term potentiation-induced cofilin translocation into spines, spine remodelling and enhancement of synaptic transmission. This mechanism has been suggested to be altered in AD, with decreased CAP2 expression solely in the hippocampus and not in the superior frontal gyrus [5]. Recent studies have shown that cofilin is required for Aβ-induced mitochondrial and synaptic dysfunction in primary neurons, and that Aβ-induced activation of cofilin represents an upstream signal impacting on tau/microtubule regulation and tauopathy [6].
Given the biological relationship between CAP2 and AD pathogenesis, we conducted a translational study to evaluate CAP2 levels in the CSF of a large sample of AD patients across different clinical stages comparing them with healthy controls and other neurodegenerative conditions and we investigated the potential correlations between CAP2 concentrations and core AD markers.
The clinical cohort included one-hundred seventy-four subjects, namely 110 patients with AD (30 prodromal and 80 mild to moderate cases), 20 with dementia with Lewy bodies (DLB), 20 with frontotemporal dementia (FTD) and 24 neurologically healthy controls. Full methodology for patients selection criteria, CAP2 measurements and analyses are reported as supplementary material.
Mean CSF CAP2 levels were significantly higher in AD patients (both prodromal to mild to moderate dementia) compared to HC, DLB and FTD patients (p=0.001) (Figure 1, A; Supplementary Table 1). In AD and HC, CAP2 levels significantly correlated with CSF t-tau (r=0.43, p<0.001), p-tau181 (r=0.57, p<0.001) levels and with CSF Aβ42 (r=-0.28, p=0.034) in sex- and age-adjusted partial correlation. In the analyses limited to AD patients and adjusted for core CSF markers, CAP2 levels showed significantly higher levels in prodromal AD compared to subjects with overt dementia (Supplementary table 1). ROC analyses were implemented to assess the discrimination accuracy of CAP2 levels in diagnosing AD compared to HC, FTD and DLB. The AUC-ROC for CAP2 CSF levels for the diagnosis of AD resulted in 0.72 (95% CI 0.64-0.80%), whereas, the CAP2/Aβ42 ratio resulted in a discrimination accuracy of 0.93 (95% CI 0.88-0.97%). The comparison between AUC-ROC curves for p-tau/Aβ42 (standard for the AD diagnosis) and CAP2/Aβ42 ratios showed no significant difference between the curves (Supplementary Figure 1).
These findings align with several recent studies showing a steady rise in biomarkers related to synapses in AD, even prior to the onset of axonal degeneration [2-3] . Furthermore, these findings are supported by the evidence of increased levels of SNAP25 in presymptomatic stages of familial AD and elevated levels of neurogranin in Aβ+ non-demented individuals compared to Aβ- subjects with an association with memory performances [1-3,7]. In agreement with other studies on different synapse-related markers, we confirmed that, compared to DLB and FTD, synaptic parameters are specifically altered in AD and likely associated to Amyloid-Precursor Protein (APP) altered metabolism since the early stages. These findings further support the view that AD is a unique form of synaptopathy different from other neurodegenerative conditions, due to the impact of APP metabolism on synaptic mechanisms [1-3] . Our results show also a significant difference of CAP2 levels between prodromal AD and mild to moderate AD. Prodromal AD showed higher CSF CAP2 levels, possibly due to aberrant spine sprouting, which is known to be a potential compensatory mechanisms highly active during early phases of the disease. In line with this, in early AD phases higher levels of few synaptic markers, such as NPTX2 have been described, whereas others markers are higher in later phases of the disease [3]. Changes in CAP2 levels may stem from diverse mechanisms. Increased levels during early Mild Cognitive Impairment (MCI) might indicate changes in gene expression or synaptic remodelling by relatively unaffected neurons in response to amyloid and neurofibrillary tangle-induced damage [8] However, disease progression could hamper the capacity of compensatory efforts, impacting on protein production, including CAP2, and leading to lower CSF levels in later stages.
In subjects with AD, CAP2 correlated with CSF p-tau181 (r=0.32, p=0.001) and t-tau (r=0.36, p<0.001) but not with CSF Aβ42, with no effect of APOE genotype on CAP2 levels (Supplementary table 2). Linear regression analyses confirmed the positive correlation of p-tau but not of t-tau levels with CSF CAP2 levels in AD subgroups (p=0.03, supplementary table 3). The potential link between CAP2 and tau pathology was thus additionally tested in neuronal cultures (Supplementary material for methodology and results details). The experiment evaluated the effect of CAP2 downregulation on total and phosphorylated tau levels in hippocampal neurons. To downregulate CAP2 we took advantage of small hairpin RNA (shRNA) packaged into recombinant adeno-associated virus (rAAV) particles and to transduce primary hippocampal neurons (Figure 1B). It was confirmed that the CAP2 protein levels were diminished in neurons expressing CAP2 shRNA (shCAP2) compared to neurons transduced with control rAAV (scrCAP2) (Figure 1C). As shown in Fig. 1D, the downregulation of CAP2 levels resulted in an increased in p-tau181 with no effect on t-tau levels.
The convergence between clinical and in vitro findings implies a plausible biological connection between CAP2 and tau abnormalities, likely mediated by cofilin [6]. In fact, down-regulation of CAP2 impairs neuronal architecture and spine shape, together with a decrease in synaptic excitatory transmission. In the hippocampal synapses of AD patients and mouse models, previous studies have demonstrated a dramatic increase in cofilin levels, along with a reduction in CAP2 synaptic availability and, accordingly, a decrease in CAP2 dimer formation at the synapse [5]. Furthermore, in AD patients’ hippocampi, cofilin precipitates a different pattern of CAP2 monomeric and dimeric forms, suggesting the presence of an ineffective CAP2/cofilin complex in hippocampal synapses that could contribute to the loss of structural plasticity of spines in AD. As a proof of concept, we demonstrated that CAP2 downregulation stimulated tau phosphorylation at Thr181, a core alteration related to AD. Furthermore, we also demonstrated that these findings were independent from the levels of total tau, which is a more aspecific marker of neuronal cell death. These findings thus further support a link between synaptic dysfunction and tau phosphorylation – possibly linked to cofilin-mediated interaction and pathology at cellular levels in AD. Given that CAP2 is an actin-binding protein and tau a microtubule-associated protein that promotes microtubule polymerization, they might influence each other via microtubule and actin networks. Morover, these findings could explain the relationship between CAP2 and p-tau levels detected in the CSF of AD patients. Several lines of evidences suggest that amyloid-induced synaptic dysfunction, maladaptive plasticity and aberrant sprouting might be among the first steps to potentially trigger tau pathology in AD models [1-3, 9,10].
Further research is needed, including the assessment of CAP2 CSF levels in subjects positive for amyloid but negative for p-tau markers, to comprehensively understand the dynamic trajectories of synaptic changes across the AD spectrum and challenge this hypotheses in vivo. Another limitation of the study was the cross-sectional design, as it did not permit the assessment of the prognostic value of CAP2 at single-subject level. Larger cohorts across AD stages are needed to validate these findings, and longitudinal studies could offer insights into CSF CAP2 trajectory. In fact, actual individual changes in the time-course of MCI towards severe AD are not discernible from our cross-sectional study, lacking multiple time points of observation. Moreover, further studies that incorporate additional CSF markers assessing synaptic integrity, microglia and copathologies [1-3,9-10] would prove beneficial in enhancing our understanding of the relationship between CAP2, synaptic function and axonal loss in vivo.
Despite limitations, the study showed that AD is characterized by CAP2 alterations since the prodromal stage and that these alterations are strongly associated with tau-related changes in vivo and in vitro, further arguing for synaptic dysfunction as central event in the pathogenesis of AD.