Authors: Jason K. Russell (1Center for Cognitive Medicine, Department of Psychiatry and Behavioral Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, USA.), Alexander C. Conley (1Center for Cognitive Medicine, Department of Psychiatry and Behavioral Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, USA.), Brian D. Boyd (1Center for Cognitive Medicine, Department of Psychiatry and Behavioral Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, USA.), J. Patrick Begnoche (1Center for Cognitive Medicine, Department of Psychiatry and Behavioral Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, USA.), Rachel Schlossberg (1Center for Cognitive Medicine, Department of Psychiatry and Behavioral Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, USA.), Allison Stranick (1Center for Cognitive Medicine, Department of Psychiatry and Behavioral Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, USA.), Adam J. Rosenberg (2Vanderbilt University Institute of Imaging Science, Vanderbilt University Medical Center, Nashville, TN 37232, USA.; 3Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, TN 37232, USA.), Lealani Mae Y Acosta (4Department of Neurology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.), Dann Martin (5Department of Clinical Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, TN 37232, USA.), Yasmeen Neal (1Center for Cognitive Medicine, Department of Psychiatry and Behavioral Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, USA.), Prabesh Kanel (6Department of Radiology, University of Michigan, Ann Arbor, MI, USA.; 7University of Michigan Morris K. Udall Center of Excellence for Parkinson's Disease Research, Ann Arbor, MI, USA.), Roger L. Albin (7University of Michigan Morris K. Udall Center of Excellence for Parkinson's Disease Research, Ann Arbor, MI, USA.; 8Department of Neurology, University of Michigan, Ann Arbor, Michigan, USA; 9Geriatric Research, Education, and Clinical Center, VAAAHS, Ann Arbor, Michigan, USA.), Michael S. Rafii (10Alzheimer's Therapeutic Research Institute, Keck School of Medicine, University of Southern California, San Diego, California, USA.), Julie Dumas (11Department of Psychiatry, University of Vermont, Burlington, VT, USA.), Paul A. Newhouse (1Center for Cognitive Medicine, Department of Psychiatry and Behavioral Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, USA.; 12Geriatric Research, Education, and Clinical Center, Veterans Affairs Tennessee Valley Health System, Nashville, Tennessee, USA.)
Categories: Article, Cholinergic, Down Syndrome, PET, [18F]-FEOBV, Aging
Source: Neurobiology of aging
Authors: Jason K. Russell, Alexander C. Conley, Brian D. Boyd, J. Patrick Begnoche, Rachel Schlossberg, Allison Stranick, Adam J. Rosenberg, Lealani Mae Y Acosta, Dann Martin, Yasmeen Neal, Prabesh Kanel, Roger L. Albin, Michael S. Rafii, Julie Dumas, Paul A. Newhouse
Adults with Down syndrome are genetically predisposed to developing Alzheimer’s disease after the age of 40. The cholinergic system, which is critical for cognitive functioning, is known to decline in Alzheimer’s disease and although first investigated in individuals with Down syndrome 40 years ago, remains relatively understudied. Existing studies suggest individuals with Down syndrome have an intact cholinergic system at birth that declines through adulthood alongside the development of Alzheimer’s disease pathology. The present study provides the first description of cholinergic terminals in vivo in non-demented adults with Down syndrome utilizing [^18^F]-fluoroethoxybenzovesamicol PET imaging. In addition, we investigated age-associated decline in cholinergic terminal density. Sixteen (16) non-demented adults with Down syndrome (mean age 35.5, 8 females) and 20 neurotypically developed individuals (mean age 35.5, 10 females) were studied, comparing radiotracer uptake groupwise and associations with age utilizing a voxel-based approach. Adults with Down syndrome displayed significantly increased [^18^F]-fluoroethoxybenzovesamicol uptake in the cerebellum, brainstem, thalamus, and numerous cortical regions compared to age-matched controls following an unpaired t-test thresholded at p<0.001 and minimum cluster size 50. Cholinergic terminal density in numerous cortical regions showed a steeper decline associated with older age in adults with Down syndrome than observed in neurotypically developed adults in the age range tested following a generalized linear model testing the interaction between age and group, thresholded at p<0.005 and minimum cluster size 50. These data suggest higher cholinergic terminal density in early adulthood in individuals with Down syndrome, with a greater age-related difference than is observed in neurotypically developed individuals.
Alterations in the cholinergic system in individuals with Down syndrome (DS) have been investigated since the early 1980s (Brooksbank et al., 1989; Casanova et al., 1985; Kish et al., 1989). The cholinergic system is vital in modulating normal cognitive function (Graef et al., 2011), and decline in this system is responsible for impairments in cognitive functioning in aging and neurodegenerative disease (Dumas & Newhouse, 2011). Disruptions in cholinergic signaling have been hypothesized to play a role in the developmental disabilities observed in DS and the age- and Alzheimer’s disease (AD)-related decline in cognition observed in adults with DS (Casanova et al., 1985; Fodale et al., 2006).
As neurotypical individuals age, the cholinergic system declines (Albin et al., 2018; M. J. Grothe et al., 2014; Kanel et al., 2022), which is associated with decreased cognitive function (Richter et al., 2014). In neurotypical individuals, age-related cholinergic decline is accelerated by the development of AD pathology. Specifically, degeneration of the basal forebrain system (Ch1–4) has been demonstrated in AD (Dumas & Newhouse, 2011; Fernández-Cabello et al., 2020; Whitehouse et al., 1981, 1982), with more recent studies suggesting the decline of this system is present in subjective cognitive decline, a risk factor for the development of mild cognitive impairment (MCI) (Nemy et al., 2023; Scheef et al., 2019). Reduced cholinergic basal forebrain volume has been associated with poorer performance on numerous cognitive endpoints in healthy older individuals and individuals with AD (Richter et al., 2014; Xia et al., 2022).
Individuals with DS are virtually certain to develop AD (Fortea et al., 2021) and are reported to display a general accelerated aging phenomenon (Zigman, 2013). The triplicated chromosome 21 contains the gene for the amyloid precursor protein (APP), with overexpression of APP believed to underlie the early widespread amyloid deposition seen in individuals with DS (Rafii et al., 2019; Wisniewski et al., 1985). The life expectancy for individuals with DS has greatly increased over the last several decades and was reported as 57 years in 2019 (Iulita et al., 2022). As such, AD is now the leading cause of death in individuals with DS (Hithersay et al., 2019), with an estimated age of AD dementia onset of 53.8 years and an estimated age of death due to AD of 58.4 years (Iulita et al., 2022). Individuals with DS have been characterized as AD stage 0 from birth (Jack et al., 2024), and the progression of AD pathology in this population is highly predictable (Fortea et al., 2021). Due to this, recent studies in populations with DS have begun using age as a surrogate for disease stage in neuroimaging biomarker studies (Boerwinkle et al., 2023; Iulita et al., 2022; Lao et al., 2024). Central cholinergic degeneration occurs early in the development of AD pathology and underlies many of the cognitive deficits observed in AD. In contrast to studies in children with DS, in adults with DS, some studies with acetylcholinesterase inhibitors have displayed modest positive effects on cognition (Heller et al., 2004; Spiridigliozzi et al., 2007), although effects are still mixed with no effects observed in young adults (Kishnani et al., 2009). Given the high risk of AD in individuals with DS, the role of basal forebrain cholinergic decline in AD symptomatology, and the accelerated aging phenomenon observed in numerous systems in AD, it is important to understand how the cholinergic system changes with aging in adults with DS.
Studies in adults with DS are relatively limited; one small study suggests a reduction in the number of neurons within the nucleus basalis of Meynert (Ch4) compared to controls, with an increased decline from 16–56 years of age in adults with DS (Casanova et al., 1985). More recent work in adults with DS, performed using magnetic resonance imaging (MRI) volumetry, indicated decreasing anteromedial (Ch1–3) and posterior (Ch4) basal forebrain volumes with the progression from asymptomatic through prodromal AD to symptomatic AD (Rozalem Aranha et al., 2023). These data indicate an important role for basal forebrain cholinergic degeneration alongside the development of AD pathology, which may underlie AD-related cognitive impairment in individuals with DS, as is observed in the typically developed population. Whether performed using MRI methodologies or post-mortem, cholinergic basal forebrain anatomy provides important information about the basal forebrain projection system in general; however, it does not indicate potential regional differences in cholinergic terminals. Assessments of age-related differences in cholinergic terminals in adults with DS have not been performed. Such studies investigating regional cholinergic terminals would provide greater insight into cholinergic decline in adults with DS compared to neurotypical individuals with AD.
The density of the central cholinergic nerve terminals can be assessed using positron emission tomography (PET) imaging. Specifically, [^18^F]-fluoroethoxybenzovesamicol ([^18^F]-FEOBV) is a vesamicol-derived radiotracer that binds to the vesicular acetylcholine transporter (VAChT) found in cholinergic nerve terminals (Mulholland et al., 1993, 1998). [^18^F]-FEOBV uptake has shown decreased binding in patients with AD (Aghourian et al., 2017), with an association between cognitive performance and [^18^F]-FEOBV uptake observed (Xia et al., 2022). Furthermore, in neurotypical individuals, regional [^18^F]-FEOBV uptake has been observed to decline with increasing age (Albin et al., 2018; Kanel et al., 2022; Okkels et al., 2023). These data suggest that [^18^F]-FEOBV PET is sensitive to differences in cholinergic terminal density seen with normal aging and in the early stages of AD.
The present study is the first to describe cholinergic terminal density in adults with DS using cholinergic PET imaging. As such, we first investigated whether individuals with DS display differences in [^18^F]-FEOBV uptake compared to an age-matched neurotypical control group. Secondly, we perform exploratory analyses to assess whether adults with DS display a greater age-associated decline in cholinergic terminal density than neurotypical controls.
Sixteen individuals with DS with an average age of 35.5 years old (range 19–50 years old, 8 male, 8 female), were recruited at Vanderbilt University Medical Center. To be considered eligible, these participants had to have a diagnosis of DS (including mosaic DS or partial trisomy 21), be aged 18–55 inclusive, with no diagnosis of dementia. Central nervous system active medications were permitted if the participant had been on a stable dose for at least 4 weeks or longer. Participants were required to have visual and auditory acuity to allow for neuropsychological testing. Exclusion criteria included any unstable medical condition or disease that could affect neuropsychological testing such as unstable cardiac problems, chronic renal failure, chronic hepatic disease, MRI contraindications or an inability to complete imaging studies, positive pregnancy test 48 hours to PET scanning for females, history of a primary or recurrent malignant disease in the last 5 years (with the exception of non-melanoma skin cancers, resected cutaneous squamous cell carcinoma in situ, basal cell carcinoma, cervical carcinoma in situ, or in situ prostate cancer with normal prostate-specific antigen post-treatment) and any abnormalities in B12 or thyroid function tests that would interfere with the study, clinically significant abnormalities on screening laboratories, previously received radiation exceeding the yearly radioactive dose of 30 mSv, and concurrent participant in a clinical trial for an investigational product or longitudinal study with overlapping measures or prohibited procedures. All TRC-DS and non-TRC-DS participants (< 25 years old) participants whose legally authorized representative signed a consent form for the Cholinergic PET Study who had completed MRI, [^18^F]-FEOBV PET, and cognitive assessment by September 2024 were included in the analysis. These participants underwent neuropsychiatric assessment (Down Syndrome Mental State Exam, and the Kaufman Brief Intelligence Test Second Edition (KBIT-2) were used to establish premorbid intellectual disability), an [^18^F]-FEOBV PET scan, and an MRI scan (see Table 1 for a summary of participant demographics and cognitive scores). Participants over 25 years old were recruited from the Trial Ready Cohort – Down Syndrome (TRC-DS), a cohort study in which participants undergo multimodal imaging and cognitive assessments (NCT04165109). The younger participants, aged 18–24 years, were recruited into a study examining cholinergic terminal density in DS (NCT05231798). The Vanderbilt University Medical Center Institutional Review Board approved both the TRC-DS and the cholinergic substudy, and participants or legally authorized representatives gave written informed consent in accordance with the Declaration of Helsinki.
The control group was selected to have the same average age and sex distribution. It consisted of 20 neurotypically developed adults with an average age of 35.5 years old (range 20–61, 10 males, 10 females). Participants under 40 were recruited at the University of Michigan, data which has been used in previous studies of [^18^F]-FEOBV binding (Albin et al., 2018; Kanel et al., 2022). To be considered eligible for this study, participants were required to have normal neurological exams, no history of neurological and psychiatric disease, and drugs that may affect cholinergic transmission were prohibited. All available participants from this study were included in the analysis. Participants over 50 were recruited at Vanderbilt University Medical Center and the University of Vermont (NCT04129060) for a separate study on central cholinergic health following menopause where a subset of participants underwent [^18^F]-FEOBV PET imaging alongside additional study procedures, including an MRI. This study recruited participants who were aged 50–70 years old, postmenopausal, non-smokers for at least the last 2 years, not taking systemic hormone therapy, no cardiovascular disease, no abnormalities in physical exam, an IQ in the normal range (>80) and normal neuropsychological testing. Participants were excluded if they had a diagnosis of MCI or dementia, a history of cancer treatment with cytotoxic or ongoing maintenance target chemotherapy, uncontrolled hypertension, uncontrolled thyroid disease, uncontrolled asthma or chronic obstructive pulmonary disease, an active peptic ulcer, uncontrolled epilepsy, contraindications to MRI or PET imaging studies. Prohibited medications included anti- or pro-cholinergic medications, cognitive-enhancing medications, and hormonal therapies. Fifty-one participants completed [^18^F]-FEOBV PET scans in this study by September 2024. Seven female participants were selected from the larger group based on age to balance the sex distribution and age of the neurotypical control and DS groups (see Table 1 for participant demographics). The Institutional Review Board at the University of Michigan approved the study containing control participants under 40 years of age, while the study containing control participants over 50 years old was approved by the University of Vermont Institutional Review Board, with the [^18^F]-FEOBV PET imaging substudy approved by the Vanderbilt University Medical Center Institutional Review Board. All participants gave written informed consent in accordance with the Declaration of Helsinki. All 13 neurotypical participants from the group under 40 years old were utilized, while seven neurotypical participants from the group over 50 years old were selected to generate a control group with a matched average age and a balanced sex distribution. Study data from the Vanderbilt University Medical Center was collected and managed using REDCap electronic data capture tools hosted at Vanderbilt University Medical Center (Harris et al., 2009, 2019).
For adults with DS, MRI scans were performed on a research-dedicated Philips 3.0T Ingenia Elition X (Philips Medical Systems, Best, the Netherlands) at Vanderbilt University Medical Center. T1-weighted scans with Repetition Time (TR) = 6.7 ms, Echo Time (TE) = 3.1 ms, and a spatial resolution of 1 x 1 x 1.2 mm^3^ (170 slices and 334-second duration) were utilized for PET image registration.
For the neurotypical individuals, MRI scans were performed on a research-dedicated Philips 3.0T Ingenia CX (Philips Medical Systems, Best, the Netherlands) at Vanderbilt University Medical Center or the University of Vermont, or a Philips 3.0T Achieva system (Philips Medical Systems, Best, the Netherlands) at the University of Michigan. T1-weighted scans with TR = 6.3 ms, TE = 2.9 ms, and a spatial resolution of 1 x 1 x 1 mm^3^ (225 slices and 338-second duration) from the scans at Vanderbilt University Medical Center and TR = 9.8 ms, TE = 4.6 ms and a spatial resolution 1 x 1 x 1 mm^3^ (136 slices) from the scans at Michigan University were utilized for PET image registration.
Participants with DS and control participants from Vanderbilt University Medical Center received 6.5 mCi (240.5 MBq) ± 10% [^18^F]-FEOBV via a slow I.V. bolus, while control participants from Michigan received 7.8 – 8.6 mCi (288.6 – 318.2 MBq) as previously described (Albin et al., 2018). Data acquisition began after a 3-hour uptake; participants underwent a 30-minute scan consisting of six 300-second frames with a voxel size of 2 mm isotropic and a field of view (FOV) of 256 mm. PET scans were performed using a Philips Vereos digital PET/CT system at Vanderbilt University Medical Center and an ECAT Exact HR+ PET tomograph (Seimens Molecular Imaging) at University of Michigan. PET images from both sites were reconstructed using the OSEM-3D method with Gaussian smoothing, 3mm full-width half maximum (FWHM). As matrix sizes were different between the two scanners, the images were subsequently resampled to match the larger voxel size prior to any further image processing. This was performed using the mri_convert command from FreeSurfer with "--voxsize 2.574 2.574 2.425" using default interpolation settings.
PET images were motion-corrected using MCFLIRT in FSL. PetSurfer (https://surfer.nmr.mgh.harvard.edu/fswiki/PetSurfer) (Greve et al., 2014, 2016) was used to coregister [^18^F]-FEOBV PET images with structural T1-weighted MRI images (to MRI space – or subject space, voxel size 1mm isotropic) and partial volume correct PET images with the region-based voxel-wise (RBV) method (Thomas et al., 2011), with smoothing to a 4mm full width at half maximum (FWHM). The PET images were normalized to the eroded supraventricular white matter, similar to previous publications with this tracer (Albin et al., 2018). The eroded supraventricular white matter reference region was generated in subject space with examples from both the adults with DS and neurotypically developed adults shown in Supplementary Figure 1 (Analysis without partial volume correction produces similar results and can be seen in Supplementary Figures 2-5). Coregistered PET and structural MRI images in subject space were imported to Advanced Neuroimaging Tools (Cullen & Avants, 2018) in Python (ANTsPy (v.0.5.3) and ANTsPyNet (v0.2.8), Python v3.12.4). Brains were extracted from structural T1-weighted MRIs using the brain extraction tool in ANTsPyNet and then transformed from subject space into MNI-152 space using symmetric normalization with ANTsPy (“SyN” function with defaults used; affine + deformable transformation, with mutual information as optimization metric). Once the T1-weighted MRI was in MNI-152 space it could be used for voxel-based morphometry (VBM) analysis, and the transformation parameters could be used to transform the PET images. PET images were transformed from subject space into MNI-152 space using the fwdtransforms function in ANTsPy, following the same symmetric normalization parameters that were generated during the co-registration of the T1-weighted MRIs from subject space to MNI-152 space.
Study-specific masks were generated using Nilearn (v0.10.4) in Python (v3.12.4) (Abraham et al., 2014). T1-weighted MRI images in MNI-152 space were used to calculate a whole brain and white matter masks. The white matter mask was then inverted to produce a whole brain mask, excluding white matter for each subject. These masks were then combined on a probabilistic level. If a voxel was within the masked area for >30% of participants, it was included in the final mask for the specific analysis. This threshold was selected empirically to ensure that the majority of grey matter voxels from participants was included.
All analysis was performed on data collected at baseline in a cross-sectional correlative approach. Voxel-based analysis was performed using Nilearn. Groupwise comparisons between adults with DS and neurotypical adults were performed using a whole-brain voxel-wise t-test. A whole-brain voxel-wise general linear model with group as the variant of interest and sex, as defined by the sex assigned at birth, and age as covariates, was also performed. An uncorrected threshold of p < 0.001 with a minimum cluster size of 50 was used to determine regions displaying a significant difference between groups. For the primary analysis, the whole-brain voxel-wise t-test, the analysis was also performed with p < 0.05 False Discovery Rate (FDR) corrected. To assess age-related change in [^18^F]-FEOBV uptake, a general linear model was used in a whole-brain voxel-wise approach with age as the variant of interest and sex as a covariate; as these endpoints were exploratory, a more permissive uncorrected p-value of p < 0.005 with a minimum cluster size of 50 was used to determine significant age-related change with both positive and negative correlations evaluated. This analysis was also performed with a significance threshold of p < 0.05, FDR corrected. To compare the rate of age-related decline between the adults with DS and neurotypically developed individuals, a general linear model assessing the interaction between group and age was performed with a p-value of p < 0.005 and cluster size 50. This analysis was also performed with a significance threshold of p < 0.05, FDR corrected. As the interaction analysis only displays significant clusters, not directionality, the beta-value for the association between age and [^18^F]-FEOBV uptake in adults with DS and the neurotypically developed age-matched control group was masked to include only negative associations. As a group x age interaction may be observed with no significant effect of age alone, no significance threshold was used, allowing the beta value to be observed in all voxels with a negative association.
VBM analysis was performed using Nilearn. To assess age-associated differences in grey matter intensity, a generalized linear model was performed with age as the variant of interest and sex as a covariate. An uncorrected p-value of p < 0.005 with a minimum cluster size of 50 was used to determine significance. The analysis was repeated with p < 0.05 FDR corrected to determine significance.
All voxel-based analyses are visualized using the xjView toolbox (https://www.alivelearn.net/xjview).
Similar to the voxel-based PET analysis, the [^18^F]-FEOBV PET image was motion corrected using MCFLIRT in FSL and coregistered to the T1-weighted MRI using PetSurfer. Cortical reconstruction and volumetric segmentation of T1-weighted MRIs was performed using the FreeSurfer imaging suite (http://surfer.nmr.mgh.harvard.edu/). These parcellations were utilized to calculate [^18^F]-FEOBV standard uptake value ratios (SUVRs) in cortical and subcortical parcellations using the eroded supratentorial white matter to calculate the normalized ratio. As these are larger volumes, PVCs were not applied.
For volumetric analysis, the volumes were calculated from the FreeSurfer parcellations in subject space, while the basal forebrain volume was calculated using the ScLimbic pipeline in FreeSurfer (Greve et al., 2021). Total intracranial volume (TIV) was calculated using a multiatlas-based approach (Huo et al., 2017).
For group-wise comparisons of SUVRs and volumes, significance in the region of interest-based analyses was determined using a student’s t-test p < 0.05 corrected for multiple comparisons using an FDR correction. A comparison of structural volumes between groups can be seen in Table 1. For [^18^F]-FEOBV and age correlations in the cortical region, a general linear model was used with [^18^F]-FEOBV SUVR as the variable of interest and age as the predictor variable. An age x group interaction term was assessed to evaluate differences in [^18^F]-FEOBV uptake with age between the two groups. All volume-based statistical analyses were performed in R-studio. Demographics were compared using a student’s t-test or chi-squared test in R studio.
Due to the group level matching approach utilized in this study, no significant differences in age or sex were observed between the neurotypically developed control group and adults with DS (Table 1). The adults with DS displayed significantly smaller volumes in the basal forebrain, hippocampus, frontal lobe, parietal lobe, temporal lobe, insular cortex, cingulate cortex, thalamus, and cerebellum, in addition to the total intracranial volume. When correcting for the smaller intracranial volume a larger occipital lobe volume was observed in adults with DS compared to neurotypically developed controls, with smaller volumes still observed in the insular cortex, cingulate cortex, and cerebellum (see Table 1 for significance levels).
Adults with DS displayed an increase in [^18^F]-FEOBV uptake across numerous brain regions when compared to age-matched neurotypically developed control participants (p < 0.001 uncorrected, minimum cluster size 50) (Figure 1). This result is also apparent when performing a GLM adjusting for age and sex (Supplementary Figure 6) and when correcting for multiple comparisons using p < 0.05 and FDR correction (Supplementary Figure 7). The increased [^18^F]-FEOBV uptake in adults with DS is most apparent throughout the cerebellum and brainstem regions. Increased uptake is also observed through numerous cortical and subcortical areas, with increased uptake bilaterally in regions of the thalamus, occipital cortex, temporal cortex, frontal cortex, insular cortex, and parietal cortex.
When performing ROI-based analyses, [^18^F]-FEOBV SUVRs displayed consistent increases across all regions examined in the adults with DS compared to neurotypical controls (hippocampus, frontal lobe, parietal lobe, occipital lobe, temporal lobe, insular cortex, cingulate cortex, thalamus, and cerebellum). All comparisons survived FDR correction (p < 0.05) (Supplementary Figure 8, See Supplementary Table 1 for significance levels).
In addition to the multiple regions of increased [^18^F]-FEOBV uptake in adults with DS, several restricted clusters display reduced [^18^F]-FEOBV uptake compared to neurotypically developed control individuals. Notably, clusters with lower [^18^F]-FEOBV binding are observed in the cingulate cortex, the parietal cortex, and the boundary of the occipital and temporal cortices.
Participants with DS display a number of clusters that have a negative association with age, i.e., older age predicts lower [^18^F]-FEOBV uptake (p < 0.005, minimum cluster size 50) (Figure 2). These effects do not persist when thresholding significance based on p < 0.05 FDR corrected. These clusters are most predominant in cortical regions, with numerous frontal, insular, parietal, cingulate, and temporal cortical clusters. A small cluster is also observed in the cerebellum. In addition to these negative associations, a small cluster in the frontal cortex displays greater [^18^F]-FEOBV uptake in older age. Voxel-based morphometry was performed to assess whether these differences were due to general grey matter loss. A small number of clusters displayed lower grey matter intensity in older age; no regions displayed high grey matter intensity (p < 0.005, minimum cluster size 50) (Supplementary Figure 9). These effects did not persist when correcting for multiple comparisons (p < 0.05, FDR corrected). Clusters displaying reductions in grey matter in older adults with DS were observed in the cerebellum, the left parahippocampal gyrus, the left temporal lobe, and the right and left frontal lobe. Of these, the only clusters that overlapped with decreased [^18^F]-FEOBV uptake was observed in the cerebellum (Supplementary Figure 10).
When performing ROI-based analyses, the insular cortex displayed a significantly lower [^18^F]-FEOBV uptake in older adults with DS (p = 0.0268), while the frontal lobe (p = 0.0532) and the cingulate cortex (p = 0.0751) displayed a statistical trend towards lower [^18^F]-FEOBV uptake in older adults with DS. None of these effects survived FDR corrections for multiple comparisons (Supplementary Table 2).
To assess whether adults with DS display a more rapid age-associated decline in cholinergic terminal density than age-matched neurotypically developed controls, an age x group interaction was assessed (p < 0.005, minimum cluster size 50) (Figure 3). These effects persisted when correcting for multiple comparisons with significance thresholded at p < 0.05, FDR corrected (Supplementary Figure 11) Adults with DS displayed a more negative age-associated decline in [^18^F]-FEOBV uptake than neurotypical controls in frontal, temporal, parietal, cingulate, insular, and occipital cortical regions. In addition to these cortical regions, a greater age-associated decline was observed in cerebellar and brainstem clusters. Small clusters in the frontal and temporal cortices displayed a more negative age-related association in the neurotypically developed group. To assist in visualizing the difference between the age-associated differences in [^18^F]-FEOBV uptake between adults with DS and neurotypically developed individuals, unthresholded whole brain beta values for the relationship between age and [^18^F]-FEOBV uptake were displayed across the whole brain for the two groups; neurotypically developed controls (Figure 4A) and adults with DS (Figure 4B). Subjectively, a range of areas display greater negative beta values in adults with DS.
When assessing the interaction between age and group using an ROI-based approach, a significant interaction was observed in the frontal lobe, parietal lobe, temporal lobe, insular cortex, and cingulate cortex. All significant regions survived corrections for multiple comparisons (Supplementary Table 3, see Supplementary Figure 12 for scatter plots displaying the interactions in regions that displayed significant interactions.
The basal forebrain cholinergic system is known to decline early in the development of AD (Daamen et al., 2023; Scheef et al., 2019; Schmitz & Nathan Spreng, 2016), and loss of the cholinergic system is associated with the cognitive deficits observed in MCI and AD (Dumas & Newhouse, 2011; Richter et al., 2022; Xia et al., 2022). Studies in individuals with DS have suggested an intact cholinergic system at birth with a decline in the basal forebrain cholinergic system during adulthood as AD-related pathology develops. Prior work has focused on the basal forebrain nuclei either at post-mortem or using MRI volumetry (Casanova et al., 1985; Rozalem Aranha et al., 2023). The present study utilizes the [^18^F]-FEOBV PET ligand in adults with DS to assess the cholinergic terminal density in cortical and subcortical regions in relation to an age-matched control group and as a function of age. This approach allows for the assessment of projections from the basal forebrain, brainstem nuclei, and cholinergic interneurons. These assessments produce two predominant findings. Firstly, adults with DS have a higher level of [^18^F]-FEOBV uptake throughout large brain areas. Secondly, adults with DS showed a greater decline in [^18^F]-FEOBV uptake across individuals of different ages than was observed in neurotypical control individuals, with the difference in [^18^F]-FEOBV PET uptake between older and younger participants being greater in adults with DS than neurotypically developed adults. These contrasting findings suggest differing mechanisms affecting the cholinergic system in adults with DS, as discussed below.
Groupwise comparisons between two age-matched groups consisting of adults with DS and neurotypically developed adults revealed higher [^18^F]-FEOBV uptake, particularly in the cerebellum and brainstem, but also observed throughout numerous cortical and thalamic regions. Interestingly, the observed increase in [^18^F]-FEOBV uptake in a range of cortical regions in adults with DS is supported by MRI basal forebrain volumetry, where previous work has revealed increased posterior basal forebrain volume (which projects throughout cortical regions) in non-demented adults with DS compared to age-matched neurotypical controls (Rozalem Aranha et al., 2023). In contrast, in autopsy studies in infants, it was found that the activity of the cholinergic enzyme acetylcholinesterase across multiple brain areas was similar between individuals with DS and non-DS controls (Brooksbank et al., 1989; Kish et al., 1989). Consistent with these data suggesting an intact cholinergic system at the beginning of life, efforts to enhance cognition in children with DS using acetylcholinesterase inhibitors have been disappointing, displaying modest or no effect on cognitive function (Kishnani et al., 2010; Spiridigliozzi et al., 2007, 2016). The increase in multiple cholinergic circuits (basal forebrain and cerebellar) suggests potential dysregulation of the cholinergic system more broadly in DS, while the data indicating normal cholinergic markers at birth suggests an upregulation of cholinergic markers during childhood by early adulthood. The reason for this higher [^18^F]-FEOBV uptake in adults with DS compared to neurotypical controls is not immediately apparent. However, this may be a response to the early development of AD pathology that is observed in adults with DS (Rafii & Santoro, 2019). Previous studies have suggested an upregulation of cholinergic markers early in the development of AD (Dekosky et al., 2002). However, many observed differences are in subcortical regions not innervated by the cholinergic basal forebrain, the primary cholinergic system affected by AD pathology in neurotypically developed individuals, and many studies indicate lower cholinergic markers as AD develops (Aghourian et al., 2021; Okkels et al., 2024; Schmitz et al., 2018; Terry & Buccafusco, 2003). An alternative hypothesis is that this elevated [^18^F]-FEOBV uptake may be an upregulation of cholinergic neurotransmission as compensation for developmental excitatory/inhibitory disturbances hypothesized in DS. SLC18A3, the gene encoding VAChT, is part of exon 1 of the choline acetyltransferase gene (CHAT) with evidence of coordinate regulation of their gene products (Gilmor et al., 1998). Increased regional VAChT density may thus indicate increased, compensatory cholinergic neurotransmission. It is important to note that although the predominant region of increased [^18^F]-FEOBV uptake in adults with DS is the cerebellum and brainstem, this is not the area of highest SLC18A3 expression (Okkels et al., 2023), which argues against a uniform and generalized upregulation of the central cholinergic system, but instead selective upregulation of SLC18A3 and so VAChT in the cerebellum and select cortical regions. Current hypotheses suggest an over-activation of the GABAergic system, resulting in excess inhibition early in development (Zorrilla de San Martin et al., 2018). One of the roles of the cholinergic system is to facilitate excitatory glutamatergic signaling in cortical (Baker et al., 2018; Marino et al., 1998) and cerebellar regions (Zhang et al., 2016); as such, the cholinergic system may display upregulation to compensate for this excess inhibition, resulting in a higher baseline level of cholinergic terminal density or signaling, as measured by [^18^F]-FEOBV uptake in adults with DS. Particularly notable increased regional cerebellar [^18^F]-FEOBV uptake was found broadly across cerebellar folia. Increased regional cerebellar [^18^F]-FEOBV uptake was seen also in early-moderate stage participants with Parkinson disease, perhaps as part of common compensatory cerebellar responses to forebrain pathologies (Brown et al., 2024 [preprint]; van der Zee et al., 2022). Previous work in non-demented adults with DS has reported increased glucose metabolic rate (GMR) measured by [^18^F]-Fluorodeoxyglucose ([^18^F]-FDG) PET during task-based conditions, in this case the increased GMR was predominant in the temporal/entorhinal cortex and hypothesized to be a compensatory mechanism for early AD pathology (Head et al., 2007).
In this cross-sectional study, adults with DS displayed lower [^18^F]-FEOBV uptake at older ages than younger adults with DS, with a greater age-associated difference than is observed in neurotypically developed individuals across a similar age range. Regions displaying lower [^18^F]-FEOBV uptake in older adults with DS within the frontal cortex and insular cortex overlap with clusters that displayed increased [^18^F]-FEOBV uptake in adults with DS when compared to neurotypically developed age-matched controls, while areas in the cingulate cortex overlapped with regions that displayed reduced [^18^F]-FEOBV uptake compared to age-matched controls. This suggests that a more rapid aging of the cholinergic system is occurring in adults with DS compared to neurotypical controls across the age range assessed while starting from a higher baseline [^18^F]-FEOBV uptake in some regions. Cholinergic terminal density has been described to decline with normal aging in the absence of neuropathology in neurotypically developed adults (Albin et al., 2018; M. Grothe et al., 2012; Kanel et al., 2022). Previous cross-sectional MRI basal forebrain volumetry studies in cognitively normal individuals have suggested a period of modest cholinergic basal forebrain volume decline in early adulthood with an acceleration in volume loss occurring from around 65–70 years of age (M. Grothe et al., 2012). The data presented here indicate that the period of relative cholinergic stability, prior to age-related decline, is reduced in adults with DS compared to neurotypically developed adults; this may be due to age-related factors and/or the development of AD pathology. A more rapid decline in the cholinergic system is observed with the development of AD pathology (Aghourian et al., 2017; Nemy et al., 2023). Individuals with DS are considered to be on the AD continuum since birth (Jack et al., 2024), with evidence of AD pathology developing at age 12 years (Lemere et al., 1996). The early and rapid development of AD pathology in adults with DS (Fortea et al., 2021; Rafii & Santoro, 2019), with amyloid PET positivity by 40 years of age and a shortened interval from amyloid positivity to accumulation of tau tangles (Zammit et al., 2021, 2024), may underly this more rapid decline in cortical cholinergic terminal density in adults with DS compared to neurotypically developed controls.
This study provides evidence for a more rapidly declining cholinergic system from a higher baseline in adults with DS compared to neurotypically developed adults. However, it is not without limitations. Firstly, this study assesses cross-sectional correlations between age and [^18^F]-FEOBV uptake. As AD-pathology develops with increasing age in adults with DS, it is not possible with this dataset to interrogate whether the observed correlations are due to age, AD pathology, or another factor. VBM analysis was performed to assess whether this decline was due to a generalized reduction in grey matter, and limited overlap between areas of reduced grey matter intensity and [^18^F]-FEOBV uptake was observed. This suggests a process selectively affecting the cholinergic system but does not preclude AD-pathology or another factor as a causative factor. Secondly, the sample size of 16 adults with DS is relatively small. However, this compares favorably to studies utilizing [^18^F]-FEOBV in studies of MCI (Xia et al., 2022), Parkinson’s disease (Horsager et al., 2022), and idiopathic REM sleep behavior disorder (Bedard et al., 2019). Somewhat larger studies have been completed with [^18^F]-FEOBV in neurotypically developed individuals (Albin et al., 2018; Kanel et al., 2022) and in Parkinson’s disease (Albin et al., 2022). Thirdly, data from two sites was utilized in this study; the neurotypically developed control group data was predominantly collected at the University of Michigan, while the data from individuals with DS was collected at Vanderbilt University Medical Center. Reconstruction methods were matched to control for site differences, and images were resampled to match the larger voxel size from the data collected at the University of Michigan. Further corrections based on site were not performed due to demographic differences (sex and condition) in the sample between sites. Finally, individuals with Down syndrome are known to display neuroanatomical differences compared to neurotypically developed individuals (Beacher et al., 2010; Pujol et al., 2018). To control for these differences, we utilized study-specific probabilistic masks, and all analyses were performed following partial volume correction and warping to a common template.
Studies are ongoing to investigate the association between AD pathology and central cholinergic terminal density to differentiate whether the observed age-related reduction in [^18^F]-FEOBV uptake in the present cohort is related to an accelerated aging phenomenon in individuals with DS or the development of AD-related pathology. These studies will utilize data from the Alzheimer’s Biomarkers Consortium – Down Syndrome (ABC-DS) and TRC-DS studies. These studies include multimodal imaging assessments, including MRI, amyloid, and tau PET imaging. Amyloid and Tau PET imaging provide measures of Alzheimer’s pathology that can be associated with measures of cholinergic terminal density, including cholinergic basal forebrain volumetry from MRI imaging and [^18^F]-FEOBV PET. The relationship between cholinergic terminal density and different measures of Alzheimer’s disease pathology will be assessed at baseline and longitudinally.
In this cross-sectional study, we have presented data indicating that adults with DS have an increased baseline cholinergic terminal density in early adulthood with a greater age-associated difference compared to neurotypical controls. We demonstrated that adults with DS display higher [^18^F]-FEOBV uptake in cortical and sub-cortical regions compared to age-matched controls. In addition, we have presented data suggesting that the cholinergic system declines faster in adults with DS than in the neurotypical population. Ongoing studies will assess whether this correlation between age and cholinergic decline is associated with the early development of AD pathology in adults with DS. Future studies will investigate the relationship between amyloid and tau pathology, cognitive function, and cholinergic decline as measured by [^18^F]-FEOBV in adults with DS. Data associating [^18^F]-FEOBV uptake with measures of AD pathology and cognitive endpoints may provide support for [^18^F]-FEOBV PET as a measure to aid in staging individuals with DS on the AD continuum and potential utility as an outcome measure in clinical trials.