Mutating PINK1 gene by paired truncated sgRNA/Cas9-D10A in Cynomolgus Monkey

Mutations of PINK1 cause early-onset Parkinson’s disease (PD) with selective neurodegeneration in humans. However,current PINK1 knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients, indicating that it is essential to generate PINK1 disease models in non-human primates (NHPs) that are highly close to humans, to investigate the unique function of PINK1 in primate brains. Paired single guide RNA (sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9, both enabling the reduction of the off-target effect without apparently compromising the on-target editing, are two optimized strategies in applying the CRISPR/Cas9 system to establish disease animal models. Here, we combined the two strategies and injected Cas9-D10A mRNA and two truncated sgRNAs into one-cell-stage cynomolgus zygotes to target PINK1 gene. We show precise and efficient gene editing of the target site in the three new born cynomolgus monkeys. The frame shift mutations of PINK1 in mutant fibroblasts leaded to the reduction of the mRNA, However, western-blot and immunofluorescencestaining confirmed the PINK1 proteinlevels werecomparable to that in the wild-type fibroblasts. We further reprogramed mutant fibroblast into induced pluripotent stem cells (iPSCs) and they show similar ability of differentiation into dopamine (DA) neurons. Taken together, our results showed that coinjection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enable the generation of human disease models in NHPs and the target editing by paired truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact the protein expression.

The effect of E-liquid exposure on Caenorhabditis elegans

E-cigarettes are being promoted as a less harmful alternative to smoking tobacco. However, vaping is a new phenomenon and safety profiles have not been fully established. Model organisms can be used to examine the cellular processes that may be changed by exposure to the E-liquids used for vaping. Mitochondria are essential in eukaryotic cells for production of ATP, protein biogenesis, metabolic pathways, cellular signalling, stress responses and apoptosis. Mitochondrial health can be used as a proxy for many aspects of healthy cellular physiology. Mutations in the PINK1 gene can lead to mitochondria-specific autophagy deficiency. We exposed two strains of Caenorhabditis elegans, CB5600 control and CC46 pink1, with 10% concentrations of nine different flavoured E liquids. We measured lifespan, movement, body size, brood size, and we examined their mitochondrial networks to investigate the effect of the E-liquids. We show that the CC46 (pink1) strain is affected by the E-liquids, even the flavours without nicotine, and that they have reduced lifespan, movement ability and mitochondrial organisation. We found that some E-liquids can dramatically shorten lifespan in this strain. Our data emphasise a need to carefully ascertain the potential harm that may be caused by the use of E-liquids.

PINK1/Parkin in Neurodegenerative Disorders

PTEN-induced kinase 1 (PINK1), a mitochondrial serine/threonine-protein kinase encoded by the PINK1 gene, is thought to protect cells from stress-induced mitochondrial dysfunction. The activity of PINK1 facilitates the binding of Parkin protein with depolarized mitochondria to induce autophagy. Mutations of PINK1causes a type of autosomal recessive early-onset Parkinson's disease. Cell depends on the surveillance systems or mechanisms like protein quality control to handle the alterations in the proteins that are induced because of these mutations. These mutant proteins are found to be pathogenic and are reported to be related to various neurodegenerative disorders. This chapter focuses on the role of PINK1/Parkin in mitochondria quality control and its subsequent effect in neurodegeneration.

In Vivo Mitochondrial Function in Idiopathic and Genetic Parkinson’s Disease

Parkinson’s disease (PD) is associated with brain mitochondrial dysfunction. High-energy phosphates (HEPs), which rely on mitochondrial functioning, may be considered potential biomarkers for PD. Phosphorus magnetic resonance spectroscopy (31P-MRS) is a suitable tool to explore in vivo cerebral energetics. We considered 10 31P-MRS studies in order to highlight the main findings about brain energetic compounds in patients affected by idiopathic PD and genetic PD. The studies investigated several brain areas such as frontal lobes, occipital lobes, temporoparietal cortex, visual cortex, midbrain, and basal ganglia. Resting-state studies reported contrasting results showing decreased as well as normal or increased HEPs levels in PD patients. Functional studies revealed abnormal PCr + βATP levels in PD subjects during the recovery phase and abnormal values at rest, during activation and recovery in one PD subject with PINK1 gene mutation suggesting that mitochondrial machinery is more impaired in PD patients with PINK1 gene mutation. PD is characterized by energetics impairment both in idiopathic PD as well as in genetic PD, suggesting that mitochondrial dysfunction underlies the disease. Studies are still sparse and sometimes contrasting, maybe due to different methodological approaches. Further studies are needed to better assess the role of mitochondria in the PD development.

Pink1 type of Early Onset Parkinson’s disease(EOPD)in Sudanese patients, 2018

Background: Parkinson’sDisease (PD) is a neurodegenerative disorder affecting the motor system. It is a chronic progressive disorder which leads to long standing disability. Objective: To study the Presentations and pink1 gene in young Sudanese patients with Parkinson’s disease . Material and Methods: A prospective study was conducted among 31 PD patients at the National center for Neurological Science (NCNS) at Khartoum state. A structured questionnaire was used for data collection. Consisted of personal data, clinical presentations and investigations. RT-PCR technique using G-spin™ kit. PINK1 gene was detected in most of the samples it was strongly positive. The data was analyzed using SPSS version 21. Results:. The majority of them 19 (61%) were located in age group 41 – 50 years; the mean age of onset was 33.4+_12 yrs. 19 (61%) of the subjects were males and 12 (39%) were female with ratio 1.6:1 (M: F), 20 (64.5%) were married. , 8 (40%) were endogamous married. 5 (62.5%) were second degree and 3 (37.5%) were third degree. 17 (85%) had children, 2 (10%) of the patient had children with Parkinson’s disease. 22 (71%) had duration more than 12 months, 12 (39%) age more than 40 years. 29 (93.5%) had tremor, 27 (87.1%) had rigidity and 23 (74.2%) had bradykinesia. 14 (45%) had positive family history of Parkinson’s disease. PINK1 gene expression was detected in 28 (90.3%) of the patients. no significant associations were found between PINK1 expression with age, gender, age at onset and family history (P> 0.05). Conclusion: This study concludes that early onset PD was common among male than female. The most affected age group was found to be 41 – 50 years and the mean age of onset 33.4yrs. Also, the patterns of the clinical features were generally similar to literature. PINK1 expression was predominant with no significant associations were found between PINK1 expression with age, gender, age at onset and family history. Key words: , Early onset,, Parkinson’s disease, Pink1 gene, Sudan.

Endosomal Rab cycles regulate Parkin-mediated mitophagy

Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson’s disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.