Microarray-based analyses of monocytes from Chinese Uygur patients with Parkinson's disease and cognitive impairment

Parkinson's disease (PD), a complex neurodegenerative disease, is characterized by the loss of dopamine neurons in the substantia nigra of the midbrain. PD patients have varying degrees of cognitive impairment (CI)1 that we term PD-CI. However, the etiology of these changes remains unexplained. We chose three Uygur male patients with PD-CI and three healthy controls of the same of nationality and sex to explore the pathogenesis of Xinjiang Uygur PD-CI using microarray-based gene expression profiling of monocytes. METHODS Subjects The cases were three Uygur men with PD-CI (sporadic) diagnosed by doctors of the First Affiliated Hospital of Xinjiang Medical University between November 2012 and January 2013. They were 65, 72, and 75 years old and were screened using the BrainBank diagnostic criteria (United Kingdom).2 When necessary, diagnosis was confirmed using a head MRI or CT scan. Secondary PD, Parkinson's syndrome, hyperthyroidism and other genetic or neural diseases were excluded. The three healthy individuals in the control group were selected from a survey population about the incidence of PD in Urumqi and were of identical gender and ethnicity as the PD patients, but were not genetically related to the patient group. Their ages were 62, 67, and 70 years. These six subjects were used for microarray-based analyzes. We also collected 31 cases of Uygur men with PD-CI (sporadic) and 35 healthy Uygur subjects for qRT-PCR verification of the microarray experiments. To investigate cognitive function, we used Mini-Mental State Examination (MMSE). MMSE scores of junior high school level ≤24 are considered to indicate cognitive disorders. This study was conducted in accordance with the Declaration of Helsinki and with approval from the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University. Written informed consent was obtained from all participants. RNA extraction Monocytes from venous blood (4.0 ml) were isolated using Histopaque-1077 Lymphocyte Separation Medium (Sigma, Louis, MO, USA) and total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA quality was determined spectrophotometrically and by using the RNA 6000 NanoChip kit with an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA), with sample acceptance limits 28S/18S ≥1.0; A260/A230 ≥1.0; A260/A280 ≥1.8. Microarray-based analysis cDNA was synthesized from 200 ng of total RNA. The cDNA was then purified using the Illumina TotalPrep RNA Amplification Kit (Ambion, Carlsbad, CA, USA) and then reverse transcribed to generate cRNA according to the manufacturer's instructions. The 1.5 μg cRNA, 10 μl RNase-free water (Ambion), and 20 μl HYB (Illumina, San Diego, CA, USA) were then added to each hybridization tube. The 30-μl sample was then added via the inlet port onto the center of each HumanHT-12 v4.0 Expression BeadChip (Illumina) and hybridization was performed at 58°C for 14–20 hours in an Illumina hybridization oven. Washes were performed in 250-ml E1BC solution (Illumina) at 55°C for 10 minutes and at room temperature for 5 minutes. BeadChip data were collected using the iScan System and BeadArray Reader and then analyzed using Illumina's GenomeStudio Gene Expression Module. This work was completed by Genergy Biotech (Shanghai, China). QuantiFast SYBR Green PCR One subset comes from 3 PD-CI and 3 healthy control subjects tested in the microarray. The other subset is a group of 31 additional Chinese Uygur patients with PD-CI and 35 healthy control subjects. Four genes, including SNCA, DNAJB4, HIPK4, and FBXW8, were selected for analysis by qRT-PCR. All primers were designed and synthesized by Genergy Biotech (Shanghai). Two-Step RT-PCR was carried out using a Funglyn FTC-3000 real-time PCR instrument (Funglyn Biotech, Toronto, Canada). Reaction Setup: 12.5 μl 2× QuantiFast SYBR Green PCR Master Mix, 2.5 μl Primer F/R and cDNA, 5.0 μl RNase-free water. Total reaction volume was 25 μl. The PCR initial activation step was 95°C, 5 minutes; denaturation was 95°C, 10 seconds; the annealing temperature for DNAJB4, HIPK4, and FBXW8 was 59°C, and for SNCA 61°C. The number of cycles was 40. Statistical analysis Background normalization was used for minimizing the amount of variation in background signals between arrays and the expected signal for unexpressed targets is equal to zero. Illumina Custom (self-developed algorithm) was used for assessing differential expression (http://supportres.illumina.com/documents/myillumina/c94519f7–9348–4308-a32f-b66ff3959e99/genomestudio_gx_module_v1.0_ug_11319121_reva.pdf). Data were analyzed by t test function in R software (Lucent Technologies, USA). Fold difference was calculated as 2-ΔΔCT. ΔΔCT(control) = CT(control) - CT(mean); ΔΔCT(case)= (CT(case) - CT(gapdh in case)) - (CT(control) - CT(gapdh in control)). A P value less than 0.05 was statistically significant. Ranksum test was used for comparing two samples 2-ΔΔCT. RESULTS MMSE assessment showed that one of the three PD-CI patients had mild cognitive disorder and that the other two cases had moderate cognitive disorder. Cluster analysis indicated that 177 genes were up-regulated and 97 were down-regulated in PD-CI subjects compared with control subjects (fold-change >2.0) (Figure 1).Figure 1.: Partial genes were up-regulated. 1–3 indicates the case group, 4–6 the control group, and red signifies over-expression of a gene.The top 10 functional pathways identified using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway maps that were affected by the observed changes in gene expression were cholinergic synapse, small cell lung cancer, transcriptional misregulation in cancer, GABAergic synapse, D-glutamine and D-glutamate metabolism, circadian entrainment, chemokine signaling pathway, glutamatergic synapse, dopaminergic synapse, arginine and proline metabolism. The most significantly affected pathway was for the cholinergic synapse (P <0.01). Up-regulation of SNCA, DNAJB and down-regulation of FBXW8 (P <0.05) was validated by qRT-PCR on the same 3 PD-CI subjects and 3 controls that were used for the microarray experiment and on another 31 PD-CI cases and 35 controls. Up-regulation of HIPK4 (P <0.05) was validated by qRT-PCR in the 3 cases and 3 controls but there was no statistically significant difference in expression levels of HIPK4 between 31 cases and 35 controls (P >0.05). DISCUSSION In this study, we identified three biomarkers (SNCA, DNAJB4, and FBXW8) that can distinguish patients with PD-CI from controls. One of the biomarkers, SNCA, which encodes alpha-synuclein, is over-expressed in peripheral monocytes of the three Chinese Uygur patients with PD-CI. Previous studies investigating the role of SNCA in cognitive impairment show that parahippocampus/transentorhinal cortex homogenates from dementia with Lewy bodies (DLB) cases accumulate significantly greater amounts of insoluble alpha-synuclein and contribute to cognitive impairment in DLB. Pathway analysis of our microarray data shows that, of the top 10 functional pathways affected by differential gene expression, four are related to synapse function: cholinergic synapse, GABAergic synapse, glutamatergic synapse, and dopaminergic synapse. The involvement of alpha-synuclein in these pathways in PD-CI is consistent with the disrupted synaptic activity seen in dopaminergic neurons of the substantia nigra and glutamatergic neurons of the hippocampus that result in global cognitive impairment.3 FBXW8 encodes an E3 ubiquitin ligase that is implicated in neuronal survival and differentiation as well as in synaptic transmission in mammalian nervous systems. The level of FBXW8 in synapses is also closely correlated with cognitive decline in Alzheimer's disease (AD). The different E3 ligase complexes can target alpha-synuclein for polyubiquitination leading to degradation by the proteosome in the endosomallysosomal pathway. In the KEGG cholinergic synapse pathway, FBXW8 has been confirmed to contribute to clearing potentially toxic hyperphosphorylated tau aggregates, the presence of which might result in a loss of cholinergic synapses and cholinergic disconnection.4 Interestingly, the dentate nucleus of the cerebellum has an extensive projection to the hippocampus, part of the brain's limbic system, indicating that it is involved in the cognitive functions of the cerebellum. When FBXW8 is up-regulated, it can significantly alleviate the abnormal accumulation of toxic proteins in the cerebellar dentate nucleus, such as insoluble ATXN2. In addition, the T allele of rs7294919, a single nucleotide polymorphism in the FBXW8 gene, is associated with lower hippocampal volume.5 Therefore, FBXW8 involvement in PD-CI is associated with the above synaptic functions, degradation of alpha-synuclein and tau, the cerebellar-hippocampal pathway, and genetic polymorphisms. DNAJB4 is a member of DNAJ/heat shock protein 40 family. Over-expression of DNAJB4 via enhancer activator protein-1 binding to promoter Yin Yang-1 and the coactivator, p300 promotes apoptosis through the JNK/JunD and caspase-3 pathway in lung cancer and AD. In this study, the second-ranking KEGG functional pathway was smallcell lung cancer. Further studies are required to determine whether the apoptosis pathway in PD-CI is similar to that in the above two diseases. The other differentially expressed gene, HIPK4, is a novel mouse homeodomain-interacting protein kinase-like gene. Over-expression of HIPK4 could contribute to the phosphorylation of p53 at serine 9, which could induce apoptosis. Although our study indicates that HIPK4 is over-expressed in Uygur PD-CI, this difference was not confirmed in the additional subjects (P >0.05). The association between HIPK4 and apoptotic mechanisms of PD-CI needs further study. Our study shows that PD-CI affects gene expression in peripheral monocytes and that SNCA, FBXW8, and DNAJB4 may be potential biomarkers of Chinese Uygur patients with PD-CI.

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