Alpha-synuclein in Parkinson's disease: molecular pathogenesis and development of genome engineering-based silencing approaches

The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Parkinsonâs disease (PD) is one such example, in which the dopaminergic neurons that manifest pathology are embedded in an inaccessible region of the midbrain, the substantia nigra pars compacta, making it unfeasible to obtain samples of the affected region from living patients. Overabundance of alpha-synuclein, encoded by the SNCA gene, has long been implicated in the pathogenesis of PD. However, the precise mechanism by which overexpression of alpha-synuclein leads to the demise of dopaminergic neurons remains elusive. Neurons differentiated from PD patient-specific iPSCs carrying multiplications of the SNCA gene may provide a means to recapitulate molecular phenotypes of the disease in vitro. The application of CRISPR/Cas9 to mammalian systems is likewise revolutionizing the utilization of genome editing in the study of molecular contributors to the pathogenesis of numerous diseases, including PD. In this body of work, I have utilized the double-nicking CRISPR/Cas9 system to mediate site-specific mutagenesis of SNCA in PD patient-specific iPSCs harboring a triplication of the SNCA gene locus, resulting in isogenic cells which can be used to phenocopy normal alpha-synuclein expression from two alleles. I have further demonstrated the utility of nuclease null or "dead" Cas9, for transcriptional silencing of alpha-synuclein expression. Finally, I have used these systems to interrogate phenotypic outcomes of SNCA triplication, demonstrating that alpha-synuclein induces endoplasmic reticulum stress and aberrant activation of a highly conserved arm of the unfolded protein response. The tools generated in these studies can be applied in future efforts to dissect the contributions of alpha-synuclein overexpression to PD pathogenesis and as a platform for the discovery of disease-modifying therapeutic approaches for PD.</p

Alpha-synuclein in Parkinson's disease: molecular pathogenesis and development of genome engineering-based silencing approaches

The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Parkinson’s disease (PD) is one such example, in which the dopaminergic neurons that manifest pathology are embedded in an inaccessible region of the midbrain, the substantia nigra pars compacta, making it unfeasible to obtain samples of the affected region from living patients. Overabundance of alpha-synuclein, encoded by the SNCA gene, has long been implicated in the pathogenesis of PD. However, the precise mechanism by which overexpression of alpha-synuclein leads to the demise of dopaminergic neurons remains elusive. Neurons differentiated from PD patient-specific iPSCs carrying multiplications of the SNCA gene may provide a means to recapitulate molecular phenotypes of the disease in vitro. The application of CRISPR/Cas9 to mammalian systems is likewise revolutionizing the utilization of genome editing in the study of molecular contributors to the pathogenesis of numerous diseases, including PD. In this body of work, I have utilized the double-nicking CRISPR/Cas9 system to mediate site-specific mutagenesis of SNCA in PD patient-specific iPSCs harboring a triplication of the SNCA gene locus, resulting in isogenic cells which can be used to phenocopy normal alpha-synuclein expression from two alleles. I have further demonstrated the utility of nuclease null or "dead" Cas9, for transcriptional silencing of alpha-synuclein expression. Finally, I have used these systems to interrogate phenotypic outcomes of SNCA triplication, demonstrating that alpha-synuclein induces endoplasmic reticulum stress and aberrant activation of a highly conserved arm of the unfolded protein response. The tools generated in these studies can be applied in future efforts to dissect the contributions of alpha-synuclein overexpression to PD pathogenesis and as a platform for the discovery of disease-modifying therapeutic approaches for PD.</p

Magnetic Resonance-Guided Focused Ultrasound Thalamotomy for Essential Tremor Under General Anesthesia: Technical Note

BACKGROUND: Magnetic resonance-guided focused ultrasound (MRgFUS) thalamotomy is an incisionless therapy for the treatment of medication-resistant essential tremor. Although its safety and efficacy has been demonstrated, MRgFUS is typically performed with the patient awake, with intraprocedural neurological assessments to guide lesioning. OBJECTIVE: To report the first case of MRgFUS thalamotomy under general anesthesia in a patient whose medical comorbidities prohibit him from being in a supine position without a secured airway. METHODS: The dentatorubrothalamic tract was directly targeted. Two sonications reaching lesional temperatures (≥54°C) were delivered without any complications. RESULTS: Lesioning was confirmed on intraoperative magnetic resonance imaging, and the patient experienced 89% improvement in his tremor postoperatively. CONCLUSION: This demonstrates the safety and feasibility of MRgFUS thalamotomy under general anesthesia without the benefit of intraprocedural neurological assessments

Alpha-Synuclein Induces the Unfolded Protein Response in Parkinson's Disease SNCA Triplication iPSC-Derived Neurons

The recent generation of induced pluripotent stem cells (iPSCs) from a patient with Parkinson's disease (PD) resulting from triplication of the α-synuclein (SNCA) gene locus allows unprecedented opportunities to explore its contribution to the molecular pathogenesis of PD. We used the double-nicking CRISPR/Cas9 system to conduct site-specific mutagenesis of SNCA in these cells, generating an isogenic iPSC line with normalized SNCA gene dosage. Comparative gene expression analysis of neuronal derivatives from these iPSCs revealed an ER stress phenotype, marked by induction of the IRE1α/XBP1 axis of the unfolded protein response (UPR) and culminating in terminal UPR activation. Neuropathological analysis of post-mortem brain tissue demonstrated that pIRE1α is expressed in PD brains within neurons containing elevated levels of α-synuclein or Lewy bodies. Having used this pair of isogenic iPSCs to define this phenotype, these cells can be further applied in UPR-targeted drug discovery towards the development of disease-modifying therapeutics

Thalamic Deep Brain Stimulation for Essential Tremor: Relation of the Dentatorubrothalamic Tract with Stimulation Parameters

BACKGROUND: In deep brain stimulation (DBS) for essential tremor, the primary target ventrointermedius (VIM) nucleus cannot be clearly visualized with structural imaging. As such, there has been much interest in the dentatorubrothalamic tract (DRTT) for target localization, but evidence for the DRTT as a putative stimulation target in tremor suppression is lacking. We evaluated proximity of the DRTT in relation to DBS stimulation parameters. METHODS: This is a retrospective analysis of 26 consecutive patients who underwent DBS with microelectrode recordings (46 leads). Fiber tracking was performed with a published deterministic technique. Clinically optimized stimulation parameters were obtained in all patients at the time of most recent follow-up (6.2 months). Volume of tissue activated (VTA) around contacts was calculated from a published model. RESULTS: Tremor severity was reduced in all treated hemispheres, with 70% improvement in the treated hand score of the Clinical Rating Scale for Tremor. At the level of the active contact (2.9 ± 2.0 mm superior to the commissural plane), the center of the DRTT was lateral to the contacts (5.1 ± 2.1 mm). The nearest fibers of the DRTT were 2.4 ± 1.7 mm from the contacts, whereas the radius of the VTA was 2.9 ± 0.7 mm. The VTA overlapped with the DRTT in 77% of active contacts. The distance from active contact to the DRTT was positively correlated with stimulation voltage requirements (Kendall τ = 0.33, P = 0.006), whereas distance to the atlas-based VIM coordinates was not. CONCLUSIONS: Active contacts in proximity to the DRTT had lower voltage requirements. Data from a large cohort provide support for the DRTT as an effective stimulation target for tremor control