Jiali Pu

Genetic variants in the RAB7L1 and SLC41A1 genes of the PARK16 locus in Chinese Parkinson's disease patients.

ABSTRACT We performed direct DNA sequencing of the RAB7L1 and SLC41A1 genes within the PARK16 locus in 205 Chinese Parkinsons disease (PD) patients. Three novel heterozygous variants were identified in SLC41A1: c.436A > G (causing p.Lys146Glu), c.1440A > G (causing p.Pro480Pro), and c.552 + 50G > A. These three variants were not present in any of the 210 genetically unrelated healthy controls of the same ethnic origin. No changes were identified in the RAB7L1 gene. Additionally, for the eight core PARK16 SNPs, no significant difference in allele or genotype frequencies was observed between PD patients and controls. Further analysis is required to determine the role of genes within the PARK16 locus in development of PD.

FERM Domain Containing Protein 7 Interacts with the Rho GDP Dissociation Inhibitor and Specifically Activates Rac1 Signaling

The FERM domain containing protein 7 gene (FRMD7) associated with the X-linked disorder idiopathic congenital nystagmus (ICN) is involved in the regulation of neurite elongation during neuronal development. Members of the Rho family of small G-proteins (Rho GTPases) are key regulators of the actin cytoskeleton and are implicated in the control of neuronal morphology. The Rho GDP dissociation inhibitor alpha, RhoGDIα, the main regulator of Rho GTPases, can form a complex with the GDP-bound form of Rho GTPases and inhibit their activation. Here, we demonstrate that the full length of the mouse FRMD7, rather than the N-terminus or the C-terminus alone, directly interacts with RhoGDIα and specifically initiates Rac1 signaling in mouse neuroblastoma cell line (neuro-2a). Moreover, we show that wild-type human FRMD7 protein is able to activate Rac1 signaling by interacting with RhoGDIα and releasing Rac1 from Rac1-RhoGDIα complex. However, two missense mutations (c.781C>G and c.886G>C) of human FRMD7 proteins weaken the ability to interact with RhoGDIα and release less Rac1, that induce the activation of Rac1 to a lesser degree; while an additional mutant, c.1003C>T, which results in a C-terminal truncated protein, almost fails to interact with RhoGDIα and to activate Rac1 signaling. Collectively, these results suggest that FRMD7 interacts with one of the Rho GTPase regulators, RhoGDIα, and activates the Rho subfamily member Rac1, which regulates reorganization of actin filaments and controls neuronal outgrowth. We predict that human mutant FRMD7 thus influences Rac1 signaling activation, which can lead to abnormal neuronal outgrowth and cause the X-linked ICN.

Plasma Exosomes Spread and Cluster Around β-Amyloid Plaques in an Animal Model of Alzheimer’s Disease

Exosomes, a type of extracellular vesicle, have been shown to be involved in many disorders, including Alzheimer’s disease (AD). Exosomes may contribute to the spread of misfolded proteins such as amyloid-β (Aβ) and α-synuclein. However, the specific diffusion process of exosomes and their final destination in brain are still unclear. In the present study, we isolated exosomes from peripheral plasma and injected them into the hippocampus of an AD mouse model, and investigated exosome diffusion. We found that injected exosomes can spread from the dentate gyrus (DG) to other regions of hippocampus and to the cortex. Exosomes targeted microglia preferentially; this phenomenon is stable and is not affected by age. In AD mice, microglia take up lower levels of exosomes. More interestingly, plasma exosomes cluster around the Aβ plaques and are engulfed by activated microglia nearby. Our data indicate that exosomes can diffuse throughout the brain and may play a role in the dynamics of amyloid deposition in AD through microglia.

Association of CD33 and MS4A cluster variants with Alzheimer's disease in East Asian populations.

CD33 and MS4A cluster variants have been identified to modulate the risk of Alzheimers disease (AD) in several recent genome-wide association studies (GWAS) in Caucasians. In the present study, we first conducted a case-control study to investigate the CD33 single nucleotide polymorphisms (SNPs) rs3865444 and rs3826656 and the MS4A cluster SNPs rs610932 and rs670139 in a cohort from eastern China that comprised 126 late-onset Alzheimers disease (LOAD) patients and 129 healthy controls. The results revealed that the frequency of rs3826656 major (G) allele carriers was higher among the LOAD patients than among the controls [P=0.005; odds ratio (OR), 1.760; 95% confidence interval (CI), 1.185-2.615]. In apolipoprotein E (APOE) ε4 allele carriers, the G allele of the SNP rs3865444 was found to be associated with an increased risk of LOAD (P=0.002; OR, 3.391; 95% CI, 1.512-7.605). Next, we re-evaluated the association between these variants and LOAD by conducting a meta-analysis using data from studies of East Asian populations, including the present case-control study, and confirmed that rs3826656 increased the risk of LOAD. In addition, we identified a significant association between rs610932 and LOAD (P=0.035; OR, 0.79; 95% CI, 0.63-0.98). Note that heterogeneity should be considered during the interpretation of these results; significant heterogeneity was identified among studies on rs3865444, even in a subgroup analysis based on stratification of studies by the country of origin. In summary, our results suggest that CD33 and MS4A cluster variants are associated with LOAD susceptibility in East Asian populations.

Essential sequence of the N-terminal cytoplasmic localization-related domain of huntingtin and its effect on huntingtin aggregates

Huntington’s disease (HD) is caused by abnormal CAG repeat expansion in the 5′-end of the Huntingtin (HTT) gene. In addition to the canonical C-terminal full-length huntingtin (htt) nuclear export signal, a cytoplasmic localization-related domain (CLRD) in the N-terminus of htt has recently been reported. Here, we analyzed this domain by introducing deletion and substitution mutations in a truncated N-terminal htt protein and subsequently monitored htt expression, aggregation and subcellular localization by immunocytochemistry and Western blot analysis. We demonstrated that Htt4–17 was the essential sequence for htt cytoplasmic localization. We also found that the subcellular distribution of htt was altered when Htt1–17 was mutated to contain amino acids of different charges, suggesting a structural requirement of Htt1–17 for the cytoplasmic localization of htt. Deletion of the first three amino acids did not affect its association with mitochondria. We observed that defective cytoplasmic localization resulted in a reduction of total htt aggregates and increased nuclear aggregates, indicating that the subcellular distribution of the protein might influence the aggregation process. These studies provide new insight into the molecular mechanism of htt aggregation in HD.

A study of six point mutation analysis of LRRK2 gene in Chinese mainland patients with Parkinson’s disease

Parkinson’s disease (PD) is a common neurodegenerative disorder and 16 genetic loci responsible for PD have been identified to date [1, 2]. Mutations in leucine-rich repeat kinase 2 (LRRK2) gene, which results in PARK8-linked PD, are common genetic causes of autosomal dominant PD. Many mutations or variants have been reported in this gene, and six of them seem to be more common: G2019S, I2012T, I2020T, and R1441C/G/H. In this study, 221 PD patients (101 females, 120 males) were recruited from the Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University of China. All patients were ethnic Han Chinese living in mainland China, and 41 patients had family history of PD. The average age at onset was 46.44 ± 18.73 (range 8–76) years for patients with family history (41 patients), and 51.89 ± 12.83 (range 9–83) years for patients without family history (180 patients). PD was diagnosed according to the criteria of the United Kingdom Parkinson’s Disease Society Brain Bank, and neurological examinations were performed by two neurologists specializing in movement disorders. A total of 120 Chinese controls from unrelated individuals were collected from the same hospital. This study was approved by the local ethics committees and written informed consent was obtained from both patients and controls. Genomic DNA was isolated from peripheral blood cells using a standard phenol–chloroform method. Mutations of G2019S, I2012T, I2020T, and R1441C/ G/H were studied. To detect R1441C/G/H mutations in exon 31 of LRRK2, PCR-restriction fragment length polymorphism assays (PCR-RFLP) were performed as described previously [3]. To detect the I2012T, I2020T, and G2019S mutations, a 331-bp fragment containing the entire exon 41 was PCR-amplified, then sequenced using forward and reverse primers; the primers were the same as those described by Lesage et al. [4]. A known synonymous mutation of Y2018Y (C6054T) was found in one patient, but not in any control. The G2019S, I2012T, I2020T, and R1441C/G/H mutations were not found in any of the PD patients or controls. This finding corresponds with the absence of G2019S, I2012T, and I2020T in 624 ethnic Chinese PD patients from Taiwan [5]. The six mutations in LRRK2 described as relevant to PD may not be associated with, or may be only a minor risk factor for, PD in Chinese patients.

A novel tyrosine hydroxylase variant in a group of Chinese patients with dopa-responsive dystonia

Dopa-responsive dystonia (DRD) comprises a heterogeneous group of movement disorders. A limited number of studies of Chinese patients with DRD have been reported. In the present study, we investigated the clinical and genetic features of 12 Chinese DRD families. Point mutation analysis of the GTP-cyclohydrolase I (GCH1), tyrosine hydroxylase (TH) and sepiapterin reductase (SPR) genes was conducted by direct sequencing. In addition, multiplex ligation-dependent probe amplification targeting GCH1 and TH was performed in “mutation-free” patients. Three reported mutations (IVS2-2A>G, c.293C>T, c.550C>T) were detected in GCH1, whereas two compound heterozygous variants were identified in TH, one of which was novel (c.1083C>A). Furthermore, this novel variant was not detected in any of the 250 ethnicity-matched, healthy controls. No exon deletions or duplicate mutations in the two genes were found in patients with DRD. No mutation in SPR was found. In addition, one patient with the IVS2-2A>G mutation in GCH1 showed signs of Parkinsonism. In conclusion, we here identified a novel heterozygous variant in TH (c.1083C>A). It is important to perform routine screening of GCH1 and TH for patients with DRD. While for patients with Parkinsonism, GCH1 mutation analysis should be performed after screening of genes like PARKIN, PARK7 (DJ-1) and PINK1.

Exosomes Secreted from HEK293-APP Swe/Ind Cells Impair the Hippocampal Neurogenesis

This study aimed to investigate the neurotoxicity of exosomes to cultured neuroblastoma and neurons in vitro and to mature and newborn neurons in the hippocampus in vivo. Recent in vitro and in vivo studies have shown that exosomes, small membranous vesicles secreted from many cell types, contain pathogenic proteins including full-length amyloid precursor protein (flAPP) and amyloid precursor protein (APP) metabolites. However, the function of these exosomes in Alzheimer disease (AD) has not been much explored. In the present study, exosomes were harvested from the conditioned medium of HEK293-APP Swe/Ind cells and injected into the hippocampal dentate gyrus region via a stereotactic method to detect their effects on the neuronal survival in vivo. These exosomes containing pathogenic proteins showed high neurotoxicity and could impair neurogenesis in the hippocampus. The data demonstrated that exosomes secreted from sick cells might damage neurogenesis and promote disease progression in AD.

Genetic analysis of ATP13A2, PLA2G6 and FBXO7 in a cohort of Chinese patients with early-onset Parkinson’s disease

Several genes have already been certified as causative genes in patients with autosomal recessive early-onset Parkinson’s syndrome with pyramidal tract signs, including ATP13A2, PLA2G6 and FBXO7. Variants in these three genes may also play roles in early-onset Parkinson’s disease (EOPD). In order to investigate the contribution of genetic variants in these three genes to Chinese sporadic EOPD patients, we screened 101 Chinese sporadic EOPD patients and 83 age- and sex-matched healthy controls using direct sequencing. Interpretation of those detected variants was performed based on the guidelines developed by the American College of Medical Genetics and Genomics (ACMG). Two missense variants, p.G360E and p.T733M, with “uncertain significance” classification were identified in the ATP13A2 gene and five synonymous variants were significantly over-represented in EOPD patients. Two missense variants, p.R53C and p.T319M, were absent in both our control group and online databases, classified as “likely pathogenic” in the PLA2G6 gene. Only benign variants were identified in the FBXO7 gene. These results indicate that rare variants of PLA2G6 may contribute to PD susceptibility in Chinese population, the ATP13A2 might be associated with higher risk for sporadic EOPD, while the FBXO7 gene doesn’t seem to be a risk factor to develop sporadic PD in Chinese population. Further biochemical and molecular biological studies needs to be conducted to support our main results in our future researches.

Recent Advances: Decoding Alzheimer’s Disease With Stem Cells

Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder that destroys cognitive functions. Recently, a number of high-profile clinical trials based on the amyloid cascade hypothesis have encountered disappointing results. The failure of these trials indicates the necessity for novel therapeutic strategies and disease models. In this review, we will describe how recent advances in stem cell technology have shed light on a novel treatment strategy and revolutionized the mechanistic investigation of AD pathogenesis. Current advances in promoting endogenous neurogenesis and transplanting exogenous stem cells from both bench research and clinical translation perspectives will be thoroughly summarized. In addition, reprogramming technology-based disease modeling, which has shown improved efficacy in recapitulating pathological features in human patients, will be discussed.