Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy.

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.

Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons.

A homozygous mutation of the DNAJC6 gene causes autosomal recessive familial type 19 of Parkinson's disease (PARK19). To test the hypothesis that PARK19 DNAJC6 mutations induce the neurodegeneration of dopaminergic cells by reducing the protein expression of functional DNAJC6 and causing DNAJC6 paucity, an in vitro PARK19 model was constructed by using shRNA-mediated gene silencing of endogenous DANJC6 in differentiated human SH-SY5Y dopaminergic neurons. shRNA targeting DNAJC6 induced the neurodegeneration of dopaminergic cells. DNAJC6 paucity reduced the level of cytosolic clathrin heavy chain and the number of lysosomes in dopaminergic neurons. A DNAJC6 paucity-induced reduction in the lysosomal number downregulated the protein level of lysosomal protease cathepsin D and impaired macroautophagy, resulting in the upregulation of pathologic α-synuclein or phospho-α-synucleinSer129 in the endoplasmic reticulum (ER) and mitochondria. The expression of α-synuclein shRNA or cathepsin D blocked the DNAJC6 deficiency-evoked degeneration of dopaminergic cells. An increase in ER α-synuclein or phospho-α-synucleinSer129 caused by DNAJC6 paucity activated ER stress, the unfolded protein response and ER stress-triggered apoptotic signaling. The lack of DNAJC6-induced upregulation of mitochondrial α-synuclein depolarized the mitochondrial membrane potential and elevated the mitochondrial level of superoxide. The DNAJC6 paucity-evoked ER stress-related apoptotic cascade, mitochondrial malfunction and oxidative stress induced the degeneration of dopaminergic neurons via activating mitochondrial pro-apoptotic signaling. In contrast with the neuroprotective function of WT DNAJC6, the PARK19 DNAJC6 mutants (Q789X or R927G) failed to attenuate the tunicamycin- or rotenone-induced upregulation of pathologic α-synuclein and stimulation of apoptotic signaling. Our data suggest that PARK19 mutation-induced DNAJC6 paucity causes the degeneration of dopaminergic neurons via downregulating protease cathepsin D and upregulating neurotoxic α-synuclein. Our results also indicate that PARK19 mutation (Q789X or R927G) impairs the DNAJC6-mediated neuroprotective function.

Gene regulation of RMR-related DNAJC6 on adipogenesis and mitochondria function in 3T3-L1 preadipocytes.

In the pilot GWAS of children obesity, DNAJC6 gene was found as a regulator for resting metabolic rate (RMR) and obesity in children aged 8-9 years. To investigate whether DNAJC6 gene regulated obesity and energy metabolism, the physiological mechanisms during adipogenesis of 3T3-L1 preadipocytes were confirmed after DNAJC6 gene was overexpressed or inhibited. Overexpressing DNAJC6 gene maintained a 3T3-L1 preadipocyte status during cell differentiation (MTT, ORO, DAPI/BODIPY). It suppressed adipogenesis and adipokine production (leptin, adiponectin), insulin signaling with IRS-GLUT4 system (RT-PCR, Western blotting), and mitochondrial function (Mito Stress Test). DNAJC6 overexpressed cells inhibited mTOR expression, but maintained LC3 expression at a high level, indicating that autophagy occurred and energy was obtained. However, when DNAJC6 gene was inhibited, fat synthesis factor was highly expressed during differentiation (PPARr, C/EBPa, aP2, etc) and the intracellular stress level increased accordingly, which affected the reduction of reserve respiratory capacity during mitochondrial respiration. Our study confirmed gene regulation of DNAJC6, overexpression or inhibition, affects adipogenesis with energy metabolism and mitochondrial functions. This basic data can be used for clinic obesity studies to control an energy imbalance.

Investigating the Endo-Lysosomal System in Major Neurocognitive Disorders Due to Alzheimer's Disease, Frontotemporal Lobar Degeneration and Lewy Body Disease: Evidence for SORL1 as a Cross-Disease Gene.

Dysfunctions in the endo-lysosomal system have been hypothesized to underlie neurodegeneration in major neurocognitive disorders due to Alzheimer's disease (AD), Frontotemporal Lobar Degeneration (FTLD), and Lewy body disease (DLB). The aim of this study is to investigate whether these diseases share genetic variability in the endo-lysosomal pathway. In AD, DLB, and FTLD patients and in controls (948 subjects), we performed a targeted sequencing of the top 50 genes belonging to the endo-lysosomal pathway. Genetic analyses revealed (i) four previously reported disease-associated variants in the SORL1 (p.N1246K, p.N371T, p.D2065V) and DNAJC6 genes (p.M133L) in AD, FTLD, and DLB, extending the previous knowledge attesting SORL1 and DNAJC6 as AD- and PD-related genes, respectively; (ii) three predicted null variants in AD patients in the SORL1 (p.R985X in early onset familial AD, p.R1207X) and PPT1 (p.R48X in early onset familial AD) genes, where loss of function is a known disease mechanism. A single variant and gene burden analysis revealed some nominally significant results of potential interest for SORL1 and DNAJC6 genes. Our data highlight that genes controlling key endo-lysosomal processes (i.e., protein sorting/transport, clathrin-coated vesicle uncoating, lysosomal enzymatic activity regulation) might be involved in AD, FTLD and DLB pathogenesis, thus suggesting an etiological link behind these diseases.

DNAJC6 Mutations Disrupt Dopamine Homeostasis in Juvenile Parkinsonism-Dystonia.

BACKGROUND: Juvenile forms of parkinsonism are rare conditions with onset of bradykinesia, tremor and rigidity before the age of 21 years. These atypical presentations commonly have a genetic aetiology, highlighting important insights into underlying pathophysiology. Genetic defects may affect key proteins of the endocytic pathway and clathrin-mediated endocytosis (CME), as in DNAJC6-related juvenile parkinsonism. OBJECTIVE: To report on a new patient cohort with juvenile-onset DNAJC6 parkinsonism-dystonia and determine the functional consequences on auxilin and dopamine homeostasis. METHODS: Twenty-five children with juvenile parkinsonism were identified from a research cohort of patients with undiagnosed pediatric movement disorders. Molecular genetic investigations included autozygosity mapping studies and whole-exome sequencing. Patient fibroblasts and CSF were analyzed for auxilin, cyclin G-associated kinase and synaptic proteins. RESULTS: We identified 6 patients harboring previously unreported, homozygous nonsense DNAJC6 mutations. All presented with neurodevelopmental delay in infancy, progressive parkinsonism, and neurological regression in childhood. 123 I-FP-CIT SPECT (DaTScan) was performed in 3 patients and demonstrated reduced or absent tracer uptake in the basal ganglia. CSF neurotransmitter analysis revealed an isolated reduction of homovanillic acid. Auxilin levels were significantly reduced in both patient fibroblasts and CSF. Cyclin G-associated kinase levels in CSF were significantly increased, whereas a number of presynaptic dopaminergic proteins were reduced. CONCLUSIONS: DNAJC6 is an emerging cause of recessive juvenile parkinsonism-dystonia. DNAJC6 encodes the cochaperone protein auxilin, involved in CME of synaptic vesicles. The observed dopamine dyshomeostasis in patients is likely to be multifactorial, secondary to auxilin deficiency and/or neurodegeneration. Increased patient CSF cyclin G-associated kinase, in tandem with reduced auxilin levels, suggests a possible compensatory role of cyclin G-associated kinase, as observed in the auxilin knockout mouse. DNAJC6 parkinsonism-dystonia should be considered as a differential diagnosis for pediatric neurotransmitter disorders associated with low homovanillic acid levels. Future research in elucidating disease pathogenesis will aid the development of better treatments for this pharmacoresistant disorder. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

DNAJC6 promotes hepatocellular carcinoma progression through induction of epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a developmental program, which is associated with hepatocellular carcinoma (HCC) development and progression. DNAJC6 (DNA/HSP40 homolog subfamily C member 6) encodes auxilin, which is responsible for juvenile Parkinsonism with phenotypic variability. However, the role of DNAJC6 in HCC development and progression is limited. Here, we report that DNAJC6 is up-regulated in HCC tissues and up-regulation of DNAJC6 expression predicts poor outcome in patients with HCC. Furthermore, overexpression of DNAJC6 enhances the ability for acquisition of mesenchymal traits, enhanced cell proliferation and invasion. DNAJC6 positively regulated expression of EMT-related transcription factor, also activating transforming growth factor ß (TGF-ß) pathway to contribute to EMT. Our findings demonstrated an important function of DNAJC6 in the progression of HCC by induction of EMT, and they implicate DNAJC6 as a marker of poor outcome in HCC.

Parkinson's disease-associated shifts between DNA methylation and DNA hydroxymethylation in human brain in PD-related genes, including PARK19 (DNAJC6) and PTPRN2 (IA-2ß).

Background: The majority of Parkinson's disease (PD) cases are due to a complex interaction between aging, genetics, and environmental factors; epigenetic mechanisms are thought to act as important mediators of these risk factors. While multiple studies to date have explored the role of DNA modifications in PD, few focus on 5-hydroxymethylcytosine (5hmC). Because 5hmC occurs at its highest levels in the brain and is thought to be particularly important in the central nervous system, particularly in the response to neurotoxicants, it is important to explore the potential role of 5hmC in PD. This study expands on our previously published epigenome-wide association study (EWAS) performed on DNA isolated from neuron-enriched nuclei from human postmortem parietal cortex from the Banner Sun Health Research Institute Brain Bank. The study aimed to identify paired changes in 5hmC and 5mC in PD in enriched neuronal nuclei isolated from PD post-mortem parietal cortex and age- and sex-matched controls. We performed oxidative bisulfite (oxBS) conversion and paired it with our previously published bisulfite (BS)-based EWAS on the same samples to identify cytosines with significant shifts between these two related epigenetic marks. Interaction differentially modified cytosines (iDMCs) were identified using our recently published mixed-effects model for co-analyzing ßmC and ßhmC data. Results: We identified 1,030 iDMCs with paired changes in 5mC and 5hmC (FDR < 0.05) that map to 695 genes, including PARK19 (DNAJC6), a familial PD gene, and PTPRN2 (IA-2), which has been previously implicated in PD in both epigenetic and mechanistic studies. The majority of iDMC-containing genes have not previously been implicated in PD and were not identified in our previous BS-based EWAS. Conclusions: These data potentially link epigenetic regulation of the PARK19 and PTPRN2 loci in the pathogenesis of idiopathic PD. In addition, iDMC-containing genes have known functions in synaptic formation and function, cell cycle and senescence, neuroinflammation, and epigenetic regulation. These data suggest that there are significant shifts between 5mC and 5hmC associated with PD in genes relevant to PD pathogenesis that are not captured by analyzing BS-based data alone or by analyzing each mark as a distinct dataset.

RMR-Related DNAJC6 Expression Suppresses Adipogenesis in 3T3-L1 Cells.

Obesity causes various complications such as type 2 diabetes, hypertension, fatty liver, cardiovascular diseases, and cancer. In a pilot GWAS study, we screened the DNAJC6 gene which is significantly related to the resting metabolic rate (RMR) in childhood obesity. With DNAJC6-overexpressed 3T3-L1 cells (TgHsp), we investigated the new obesity mechanism caused by an energy imbalance. After differentiation, lipid droplets (Oil red O staining) were not formed in TgHsp cells compared to the control. TgHsp preadipocyte fibroblast morphology was also not clearly observed in the cell morphology assay (DAPI/BODIPY). In TgHsp cells, the expression of PPARγ, C/EBPα, and aP2 (adipogenesis-related biomarkers) decreased 3-, 39-, and 200-fold, respectively. The expression of the adipokines leptin and adiponectin from adipose tissues also decreased 2.4- and 840-fold, respectively. In addition, the levels of pHSL(Ser563) and free glycerol, which are involved in lipolysis, were significantly lower in TgHsp cells than in the control. The reduction in insulin receptor expression in TgHsp cells suppressed insulin signaling systems such as AKT phosphorylation, and GLUT4 expression. Degradation of IRS-1 in 3T3-L1 adipocytes was caused by chronic exposure to insulin, but not TgHsp. Mitochondrial functions such as oxygen consumption and ATP production, as well as proton leak and UCP1 protein expression, decreased in TgHsp cells compared to the control. Moreover, autophagy was observed by increasing autophagosomal proteins, LC3, on Day 8 of differentiation in TgHsp cells. Through our first report on the DNAJC6 gene related to RMR, we found a new mechanism related to energy metabolism in obesity. DNAJC6 expression positively suppressed adipogenesis, leading to the subsequent resistance of lipolysis, adipokine expression, insulin signaling, and mitochondrial functions.

Clinical features and genetic analysis of SGCE myoclonus-dystonia: A case report.

Myoclonus-dystonia caused by mutations in the SGCE gene is clinically characterized by early onset, myoclonus, and dystonia. Here we describe a family in which several members exhibit varying degrees of myoclonus and dystonia, caused by a novel heterozygous mutation in the SGCE gene.

DNAJC6 mutation causing cranial-onset dystonia with tremor dominant levodopa non-responsive parkinsonism: A novel phenotype.

DNAJC6 mutation causes two types of phenotypes: slowly progressive parkinsonism with levodopa response and rapidly progressive parkinsonism with additional manifestations like intellectual disability, epilepsy etc. We report a new phenotype wherein an adolescent girl developed blepharospasm followed by jaw opening, lingual and cervical dystonia followed by tremors of limbs (rest and action) with rigidity, bradykinesia. The dystonia-parkinsonism phenotype has not been described. She had novel homozygous missense mutation in DNAJC6 gene.

Defining the Dynamic Regulation of O-GlcNAc Proteome in the Mouse Cortex---the O-GlcNAcylation of Synaptic and Trafficking Proteins Related to Neurodegenerative Diseases.

O-linked conjugation of ß-N-acetyl-glucosamine (O-GlcNAc) to serine and threonine residues is a post-translational modification process that senses nutrient availability and cellular stress and regulates diverse biological processes that are involved in neurodegenerative diseases and provide potential targets for therapeutics development. However, very little is known of the networks involved in the brain that are responsive to changes in the O-GlcNAc proteome. Pharmacological increase of protein O-GlcNAcylation by Thiamet G (TG) has been shown to decrease tau phosphorylation and neurotoxicity, and proposed as a therapy in Alzheimer's disease (AD). However, acute TG exposure impairs learning and memory, and protein O-GlcNAcylation is increased in the aging rat brain and in Parkinson's disease (PD) brains. To define the cortical O-GlcNAc proteome that responds to TG, we injected young adult mice with either saline or TG and performed mass spectrometry analysis for detection of O-GlcNAcylated peptides. This approach identified 506 unique peptides corresponding to 278 proteins that are O-GlcNAcylated. Of the 506 unique peptides, 85 peptides are elevated by > 1.5 fold in O-GlcNAcylation levels in response to TG. Using pathway analyses, we found TG-dependent enrichment of O-GlcNAcylated synaptic proteins, trafficking, Notch/Wnt signaling, HDAC signaling, and circadian clock proteins. Significant changes in the O-GlcNAcylation of DNAJC6/AUXI, and PICALM, proteins that are risk factors for PD and/or AD respectively, were detected. We compared our study with two key prior O-GlcNAc proteome studies using mouse cerebral tissue and human AD brains. Among those identified to be increased by TG, 15 are also identified to be increased in human AD brains compared to control, including those involved in cytoskeleton, autophagy, chromatin organization and mitochondrial dysfunction. These studies provide insights regarding neurodegenerative diseases therapeutic targets.

Levodopa responsive-generalized dystonic spells and moaning in DNAJC6 related Juvenile Parkinson's disease.

This is a case of young man who has severe generalized dystonic spells with moaning with severe parkinsonism, which improves with dopaminergic medications. Patient was found to be DNAJC6 related juvenile Parkinson's disease. This case adds to the phenotypic variability of the DNAJC6 juvenile Parkinson's disease as severe dystonic spells and moaning has not been reported previously. Early diagnosis and symptomatic treatment with dopaminergic medications helps significantly to improve the quality of life.

Haplotype Analysis on the Relationship of the DNAJC6 Gene with Early-Onset Parkinson's Disease Risk in a Chinese Population.

BACKGROUND: DNAJC6 gene is one of the Parkinson's disease (PD) related genes, but relationship between DNAJC6 polymorphisms and PD remains unclear. OBJECTIVE: We aims to examine the association between genetic variations in DNAJC6 gene and idiopathic early-onset PD (EOPD) in the Chinese population. METHODS: Exons and intron/exon boundaries of DNAJC6 gene was amplified and sequenced in 135 EOPD patients and 212 healthy controls. Single nucleotide polymorphisms (SNP)-based and haplotype-based association study between EOPD and DNAJC6 was conducted. SNP-SNP interactions were investigated using the generalized multifactor dimensionality reduction (GMDR) method. We further evaluated the effect of variants on gene function and expression using online in silico algorithms and databases. RESULTS: We found fourteen previously reported SNPs in the DNAJC6 gene. The frequencies of variant alleles in rs11208644, rs4582839 and rs4915691 were observed significantly higher in EOPD group compared to healthy controls, while in rs6588144 was significantly lower. Additionally, haplotype analysis indicated that the CTCACTCGGC, CTTACTCGGC and TTTGTTCGAC haplotypes were associated with higher EOPD risk in EOPD patients. SNP-SNP interaction analysis showed that rs12077111-rs4592284 SNP combination was the best model with higher EOPD risk. Based on the in silico analysis results, these SNPs were predicted to be no harm to the protein function, but might lead to possible changes in splice site and alter the expression level of DNAJC6. CONCLUSION: Our study indicated that EOPD was associated with several SNPs and haplotypes of DNAJC6 gene.

Other Proteins Involved in Parkinson's Disease and Related Disorders.

In order to explain the molecular causes of Parkinson's Disease (PD) it is important to understand the effect that mutations described as causative of the disease have at the functional level. In this special issue, several authors have been reviewing the effects in PD and other parkinsonisms of mutations described in LRRK2, α-synuclein, PINK1-Parkin-DJ-1, UCHL1, ATP13A2, GBA, VPS35, FBOX7 and HTRA2. In this review, we compile the knowledge about other proteins with a more general role in neurodegenerative diseases (MAPT) or for which less data is available due to its recent discovery (EIF4G1, DNAJC13), the lack of structural or functional data (as for PLA2G6 or DNAJC6), or even their doubtful association with the disease (as for GIGYF2, SYNJ1 and SPR). Also the cellular pathways involved in this disease are reviewed, with the goal of having an overview of the effects on the proteins and its possible role in the disease. This knowledge could also serve as the basis for designing tools that may potentially be used as a treatment for the disease, such as inhibitory or activating molecules, as well as other involved in regulating the half-life of the proteins involved.

DNAJC6 mutations are not common causes of early onset Parkinson's disease in Chinese Han population.

DNAJC6 has been reported as a causative gene for early onset Parkinson's disease (EOPD) in some populations, and different mutations have been reported to be associated with EOPD. However, Until now, there is limited information about DNAJC6 gene test in sporadic EOPD patients in Chinese population. Herein, we performed comprehensive DNAJC6 mutation screenings in 117 EOPD patients from Chinese population. None of the reported disease-causing mutations were found. However, we identified a novel non-synonymous heterozygous variant c.2798T>C (p.Val933Ala). Bioinformatics analysis demonstrate that the c.2798T>C variant exhibits highly conserved residues across species. Our data suggests that DNAJC6 mutations are not common causes of EOPD in Chinese population.

Comparative deletion mapping at 1p31.3-p32.2 implies NFIA responsible for intellectual disability coupled with macrocephaly and the presence of several other genes for syndromic intellectual disability.

BACKGROUND: While chromosome 1 is the largest chromosome in the human genome, less than two dozen cases of interstitial microdeletions in the short arm have been documented. More than half of the 1p microdeletion cases were reported in the pre-microarray era and as a result, the proximal and distal boundaries containing the exact number of genes involved in the microdeletions have not been clearly defined. RESULTS: We revisited a previous case of a 10-year old female patient with a 1p32.1p32.3 microdeletion displaying syndromic intellectual disability. We performed microarray analysis as well as qPCR to define the proximal and distal deletion breakpoints and revised the karyotype from 1p32.1p32.3 to 1p31.3p32.2. The deleted chromosomal region contains at least 35 genes including NFIA. Comparative deletion mapping shows that this region can be dissected into five chromosomal segments containing at least six candidate genes (DAB1, HOOK1, NFIA, DOCK7, DNAJC6, and PDE4B) most likely responsible for syndromic intellectual disability, which was corroborated by their reduced transcript levels in RT-qPCR. Importantly, one patient with an intragenic microdeletion within NFIA and an additional patient with a balanced translocation disrupting NFIA display intellectual disability coupled with macrocephaly. CONCLUSION: We propose NFIA is responsible for intellectual disability coupled with macrocephaly, and microdeletions at 1p31.3p32.2 constitute a contiguous gene syndrome with several genes contributing to syndromic intellectual disability.

Genetics of Parkinson's disease--state of the art, 2013.

In the past 15 years there has been substantial progress in our understanding of the genetics of Parkinson's disease (PD). Highly-penetrant mutations in different genes (SNCA, LRRK2, VPS35, Parkin, PINK1, and DJ-1) are known to cause rare monogenic forms of the disease. Furthermore, different variants with incomplete penetrance in the LRRK2 and the GBA gene are strong risk factors for PD, and are especially prevalent in some populations. Last, common variants of small effect size, modulating the risk for PD, have been identified by genome-wide association studies in more than 20 chromosomal loci. Here, I first outline the evolution of the research strategies to find PD-related genes, and then focus on recent advances in the field of the monogenic forms, including VPS35 mutations in autosomal dominant PD, and DNAJC6 and SYNJ1 mutations in recessive forms of juvenile parkinsonism. Additional genetic determinants of PD likely remain to be identified, as the currently known mutations and variants only explain a minor fraction of the disease burden. There is great expectation that the new DNA sequencing technologies (exome and whole-genome sequencing) will bring us closer to the full resolution of the genetic landscape of PD.