Abnormal resting‐state functional connectivity in posterior cingulate cortex of Parkinson's disease with mild cognitive impairment and dementia

To investigate changes in the functional connectivity (FC) pattern in the posterior cingulate cortex (PCC) of Parkinson's disease (PD) patients with mild cognitive impairment and dementia by employing resting‐state functional magnetic resonance imaging (RS‐fMRI).

PD with mild cognitive impairment (PD-MCI), a well-defined "transition state" between PD with no cognitive impairment (PD-NCI) and PDD, is an early manifestation of dementia and indicates an increasing risk of PDD. 5 Approximately 25% of newly diagnosed patients with PD satisfy the clinical criteria of PD-MCI, which affects a number of cognitive capacities, such as memory, visual-spatial function, and attention/executive ability. 6 In a 3-year follow-up study, more than 25% of PD-MCI patients, in comparison with less than 1% of PD-NCI patients, developed dementia. 5 Hence, early screening of PD-MCI patients and early intervention will one day be important for delaying the progression to PDD.
Resting-state functional MRI (RS-fMRI), a new noninvasive method for assessing functional brain connectivity at rest, has located a series of intrinsic connectivity networks (ICNs) in the brain, including the default mode network (DMN), the dorsal attention network (DAN), and the frontoparietal networks (FPN). All these networks are dynamically interrelated and vital for cognitive processing. 7 Using RS-FMRI, Hacker et al have found that striatal functional connectivity in the Parkinson's disease group was markedly altered. 8 Other previous studies have documented a functional disruption of the DMN in nondemented patients with PD, specifically between posterior cingulate cortex (PCC), medial prefrontal cortex, and inferior parietal nodes, suggesting that a dysfunction in the DMN connectivity may promote the development of cognitive decline in PD. 9 Gorges et al have reported that along with PD-related pathological progression, functional disruptions within the DMN seem to be strongly associated with cognitive impairment. 10 During cognitive processing, PCC, which plays a crucial role in DMN, is functionally related to ventral anterior cingulate cortex (vACC) and the DMN-related brain regions. 11 Previous studies have found white matter lesions 12 or reduced metabolism 13 in the PCC of PDD patients when compared with nondemented PD patients. Therefore, changes in the functional connectivity (FC) in the PCC might be implicated in the cognitive impairment of patients with PD. To our best knowledge, resting-state functional connectivity in PD patients with cognitive impairment (PD-CI) has been scarcely investigated in the available literature. Thus, the current case-control study attempted to explore the patterns of FC in the PCC of PD-MCI and PDD patients by RS-fMRI.

| Neuropsychological measurements
A senior neuropsychologist administered the tests to HC subjects and patients who were in the "on" medication state. The motor state was evaluated using the Hoehn and Yahr scale and the motor section of the unified Parkinson's Disease Rating Scale (UPDRS-III). The depression severity in PD patients and controls was assessed with Hamilton Depression Scale (HAMD) to eliminate subjects with severe depression (HAMD scores >20). Drug intake was recorded, and dopaminergic treatment was calcu-  15 The diagnosis and differential diagnosis of patients as PD-MCI or PDD were made accordingly. 15,16 PD-MCI patients were diagnosed by the level I category guidelines of the Movement Disorder Society, 15 and patients not satisfying the criteria for PD-MCI or PDD were diagnosed as PD-NCI.

| MRI acquisition
MRI images were obtained on a 3.0 T GE Medical System scanner equipped with an 8-channel parallel head coil. During the resting-state scan, participants lay on the back with their heads held in a foam padding and rubber earplugs to reduce noise. They were instructed to breathe calmly and not to think or fall asleep.
All DICOM files obtained from the scanner were converted into Neuroimaging Informatics Technology Initiative (NIfTI) file format.
For each participant, the first 10 time points were discarded for signal equilibrium and participants' adaptation to the scanning noise (each subject had 230 useful sets of volumes). Next, functional images received the following preprocessing steps: slice timing, motion correction, spatial standardization to the Montreal Neurological Institute (MNI) EPI template (resampling voxel size = 3 × 3 × 3 mm 3 ) in SPM8, and spatial smoothing with an 8-mm full-width at halfmaximum (FWHM) Gaussian kernel. Linear trend removal and temporal band-pass filtering (0.01-0.08 Hz) were performed on the time series of each voxel. Further analysis only included subjects with less than 1.5-mm maximum displacement in the x-, y-, or z-plane, and less than 1.5° of angular rotation on each axis.

| Functional connectivity and statistical analysis
Seed-based correlation was analyzed to identify increased or decreased cerebellar FC with REST software. Regions of interest (ROIs) were set at the bilateral PCC according to the automated anatomical labeling (AAL) template. The averaged time course was extracted from the bilateral PCC, and the correlation was then analyzed in a voxelwise manner to generate the FC in the PCC by computing the temporal cross-correlation between the mean time series of each ROI and the time series of each voxel within the brain. Finally, the correlation coefficient map was converted into Z-scores by Fisher's r-to-z transform to improve the normality, thus acquiring the entire brain Z-score map of each subject.
One-sample t tests were respectively performed on the Zscore map of each of the four groups. Within-group multiple comparisons were used to identify the FC networks in PCC of the four groups, with age and gender as covariates. Pearson's correlation was used to calculate the correlation between the intensity of FC in the PCC and the MoCA scores of PD groups. Significant differences were set at a corrected significance level of P < 0.05.
The threshold correction was performed with AlphaSim program in REST software.
Statistical Package for Social Sciences (SPSS, version 19.0) was used for statistical analysis. One-way analysis of variance (ANOVA) was adopted for comparisons between demographic factors across groups, the independent-samples t test for comparisons between groups, and Pearson's chi-squared test for comparisons between categorical variables (hand dominance and sex). The statistical significance threshold was set at P < 0.05.

| Demographics and global cognition
A total of 36 subjects were enrolled in this study, including nine PD-NCI patients, nine PD-MCI patients, nine PDD patients, and nine matched healthy subjects. Their demographic and clinical features are summarized in Table 1. The patients with PD bore a great similarity in disease duration, sex ratio, length of education, severity of depressive symptoms, handedness, UPDRS-III motor score, and PD medications. Compared with healthy controls and the PD-NCI group, the PD-MCI group performed more poorly in MMSE (P < 0.05) and MoCA tests (P < 0.01), and the PDD group had lower MMSE (P < 0.01) and MoCA scores (P < 0.01). PDD, PD with dementia; ANOVA, one-way analysis of variance. *Indicates group differences at a significance level of P < 0.05 when compared with HC group/PD-NCI group. **Indicates group differences at a significance level of P < 0.01 when compared with HC group/PD-NCI group. † Indicates group differences at a significance level of P < 0.05 when compared with PD-MCI group. † † Indicates group differences at a significance level of P < 0.01 when compared with PD-MCI group.

| Single domains cognition
Compared with the PD-NCI group, the PD-MCI group fared poorly in the assessment of memory with WAIS-IV Logical Memory subtest, the evaluation of visuospatial function with Benton's Judgment of Line Orientation and Hooper Visual Organization test, and the assessment of language with WAIS-IV Similarities test (P < 0.05) ( Table 2).

| PCC connectivity: within-group analyses
Between-group comparison showed different patterns of functional connectivity in the PCC of the HC group and PD groups.
P < 0.05 was set as the threshold for display (cluster size: >54 voxels; AlphaSim corrected). Blue and green areas represent regions showing significantly decreased connectivity. The color bar indicates the T value from two-sample t test analysis between the two groups.

| PCC connectivity between the PD-NCI group and the HC group
Compared with the HC group, the PD-NCI group reported markedly decreased connectivity between the PCC and several brain areas, including the left superior frontal gyrus, right angular gyrus, superior parietal gyrus. No regions were found showing increased connectivity to the PCC of the PD-NCI group (Table 3A and Figure 1A).  (Table 3B and Figure 1B).  (Table 3C and Figure 1C).

| PCC connectivity between the PDD group and the PD-MCI group
Compared with the PD-MCI group, the PDD group showed increased PCC functional connectivity to the left caudate, right thalamus, precuneus, middle frontal gyrus, and right angular gyrus. No regions indicated a markedly decreased connectivity to the PCC of the PD-MCI group (Table 3D and Figure 1D).

| Correlation between PCC connectivity and MoCA scores in the PD-MCI group and the PD-NCI group
A positive correlation was found between the MoCA scores and the strength of PCC connectivity with the angular gyrus and posterior cerebellum, while a negative correlation was evident between the scores and the strength of functional connectivity of PCC with the primary visual cortex, cuneus, lingual gyrus, and precuneus.
( Table 3E and Figure 2A,B). All values were expressed as mean ± SD. *Indicates group differences at a significance level of P < 0.05 when compared with PD-NCI group. **Indicates group differences at a significance level of P < 0.01 when compared with PD-NCI group.

| D ISCUSS I ON
In present study, we found that the presence of cognitive impairment in PD was associated with abnormal resting-state FC connectivity patterns in the PCC in PD-MCI and PDD patients and that an increased FC strength between the PCC and certain re-

| Increased functional connectivity in PD-MCI patients
With the application of RS-fMRI, studies have found that PD-NCI patients, who transition more quickly into PD-MCI (<1 year), reported decreased resting-state functional connectivity mainly between the medial prefrontal cortex and the posterior cingulate cortex and between the parieto-occipital areas and the caudate. 17 Another study has found markedly reduced FC in the PCC of the right parahippocampus of the PDD patients and documented that the FC strength of the PCC with the right MTL in PD was noticeably correlated with MoCA scores by insights from both functional and anatomical connections (DTI). 18 However, the dynamic changes with the progression of cognitive impairment were not observed. The current study assessed resting-state functional connectivity of the posterior TA B L E 3 Difference of PCC FC in patients between HC group, PD-NCI group, PD-MCI group, and PDD group, and the correlation between PCC connectivity and MoCA scores in PD-MCI group and PD-NCI group  The prefrontal cortex has been shown to play essential roles in executive functions. 19 The temporal lobe, which includes the middle temporal gyrus and hippocampus as its constituents, is mainly involved in visual perception, sensory information processing, memory, speech comprehension, and emotional activity. It also serves as a common semantic network of words and images, from the upper left occipital brain back through the medial temporal cortex to the inferior frontal gyrus. 20 The relationship between the cerebellum and cognitive function is also widely acknowledged. The cerebellum participates in advanced cognitive function mainly through the cortexthalamus-cerebellar loops, of which the posterior lobe is involved in memory and executive function. 21 The increased functional connectivity in the PCC with such regions reported in PD-MCI patients may not participate in the poor performance of PD-MCI patients when they were compared with PD-NCI patients. According to the functional imaging of healthy subjects, precuneus plays a central role in a series of highly integrated tasks including visuospatial imagery, episodic memory retrieval, and self-processing operations, namely first-person perspective taking and an experience of agency. 22 Subregions with different functions in the angular gyrus (AnG) help to restore episodic and semantic memories. 23 The increased FC observed in those regions may be a resource recruitment as an initial response to the mild cognitive dysfunction in PD patients.

| Decreased functional connectivity in PDD patients
With the progression of cognitive dysfunction, this increased FC observed in the PD-MCI patients was gradually lost in the PDD group.
Moreover, a decreased functional connectivity between the PCC and the caudate or thalamus was observed ( Figure 3C 21 Compared with other regional activity measurements, the detection of abnormal functional connectivity in the cerebellum can be a more sensitive and preferred index for functional disturbance in aMCI patients. 28 In our study, the FC strength of the PCC with the thalamus did not decrease/ increase in PD-MCI patients, indicating that it may be retained in PD-MCI, instead of being a compensatory node. Because of this, we speculate that the "prefrontal cortical-cerebellar loop" may act as a compensatory loop in PD-MCI patients, and degeneration of functional connectivity in this loop may be an important mechanism for the progression of cognitive dysfunction ( Figure 3D). A recent study has claimed that a decline in episodic memory may not be adequate to assess cognitive decline in PD patients and that PD-MCI patients may display an early decline in working memory and visuospatial processing before a clinical declaration of PDD. 29 Therefore, functional connectivity between the PCC and other regions such as middle frontal gyrus, right thalamus, and angular gyrus is also involved in the progression of PD-CI.

| Neuropsychology abnormalities in PD-MCI patients
MoCA test has been recommended for screening PD-MCI patients, with an excellent sensitivity of 89-93.1% and a good specificity of 84%. 30 MoCA score <26 is proposed as level I criterion for PD-MCI diagnosis by Movement Disorder Society Task Force. 15 However, MoCA is believed to be limited in diagnostic accuracy for PD-MCI. 31 Therefore, we further detected cognitive function of multiple cognitive domains in the PD-MCI and PD-NCI group using neuropsychological test battery recommended by Movement Disorder Society Task Force. 15 In line with MoCA evaluation results, the PD-MCI group showed impairments in the aforementioned cognitive domains, among which the logical memory function was significantly damaged. This is consistent with the findings of previous studies which document that PD patients demonstrate cognitive decline in the related cerebral regions. 6,32,33

| CON CLUS ION
In summary, resting-state functional connectivity in the PCC is as-

| Limitations and outlook
However, our research has some limitations. First, only a small number of subjects were enrolled in the current study, and the diagnosis of PC-MCI patients only satisfied the level I diagnostic criterion.
Second, due to motor dysfunction and poor cooperation, comprehensive cognitive function assessment was not carried out in PDD patients and so no more detailed and comprehensive understanding of the characteristics and progression of cognitive function of PD patients was obtained. Third, the potential impact of different medication protocols was not ruled out in our study. However, no significant difference in the amount of levodopa was found between the PD groups. Future studies will recruit more PD patients with a follow-up design to verify our hypotheses. We are going to explore imaging markers of PD-MCI combining different imaging techniques such as RS-fMRI and DTI method.

ACK N OWLED G M ENTS
We would like to thank Professor Hongzhi Huang from School of Foreign Languages of Fujian Medical University for his kind proofreading and polishing this manuscript.

CO N FLI C T O F I NTE R E S T
On behalf of all authors, the corresponding author states that there is no conflict of interest.

E TH I C A L S TA N DA R DS
The study protocol was approved by the ethical committee for med-