Poster BB8 - Living Deep Brain Stimulation for External Control of Striatal Dopamine

Introduction: Parkinson’s Disease (PD) is a chronic multifactorial neurodegenerative disease primarily recognized by its hallmark clinical presentation of resting tremor, bradykinesia, rigidity, and postural instability[1]–[3]. These symptoms result from the loss of more than 30%-70% of dopaminergic neurons in the substantia nigra pars compacta (SNpc) due to the presence of cytoplasmic alpha-synuclein aggregates (Lewy bodies) and “dystrophic neurites” (Lewy neurites). This leads to the loss of innervation of the striatum through the nigrostriatal pathway and severely diminished levels of dopamine in the striatum[2], [3]. Current gold standard treatments for PD, such as dopamine replacement therapy and deep brain stimulation (DBS), gradually lose efficacy, are nonspecific, and do not replace lost dopaminergic neurons in the brain. Additionally, DBS utilizes inorganic microelectrodes that can cause neuronal loss, glial scarring, and inflammation. We are advancing an alternative strategy to control striatal dopamine levels featuring implantable, living axonal tracts projecting from a discrete population of optically controlled dopaminergic neurons referred to as Dopaminergic Tissue Engineered Living Electrodes (dTELEs) (Fig. 1). The dTELEs consist of aggregated dopaminergic neurons and long-distance axonal tracts enclosed within hydrogel microcolumns. We have previously shown that "living electrodes" fabricated using primary rat neurons can be externally controlled using light after being transduced to express channelrhodopsins and can establish connections with existing neural circuitry after implantation into rat brains[4], [5]. Our current objective is to optimize the transduction efficiency of human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons using adeno-associated viral (AAV) vectors to allow for optical control while maintaining cell health.

Materials and

Methods: iPSC-derived dopaminergic neurons were incubated for 24 or 48 hours during the aggregation process using multiple titers of various AAV vectors to express ChR2 or ChrimsonR, each with a synapsin promoter and a fluorescent reporter. Following incubation, aggregates were seeded into a hydrogel microcolumn (1000 µm outer diameter, 500 µm inner diameter, 4-6mm length, and 110,000 neurons) (dTELEs) or plated on a 2D surface. Phase-contrast microscopy images were taken at multiple time points up to 35 days in vitro (DIV) to evaluate neuronal health and axonal extension within the microcolumns. At terminal time points, cultures were fixed for immunocytochemistry (ICC) to determine vector transduction and phenotype or underwent a standard viability assay to ascertain health and morphology. Using ICC and confocal microscopy, the aggregates were confirmed to be dopaminergic neurons through the co-expression of tyrosine hydroxylase (TH) and b-tubulin III (Tuj1).

Results, Conclusions, and Discussions:

Results: Aggregates seeded in dTELEs or plated on a 2D surface were cultured successfully, with axons in dTELEs projecting throughout the full length of the hydrogel microcolumns. Higher transduction efficiency was achieved in groups incubated with vectors for 48 hours, treated with fluorophore-only vector, or that received the highest vector titers as indicated by the fluorescent reporter intensity and extent of coverage throughout the neuronal somata and axonal tracts (Fig. 2). However, cell viability and health metrics decreased with increases in the viral titer in some groups, and differential health metrics were found across the various vectors at matched titers. Ongoing studies compare the evoked dopamine release in dTELEs induced by electrical and optical stimulation to further assess the vector transduction efficiency.

Conclusion: Even after the passing of over 200 years since PD was first described by James Parkinson in 1817, current treatment options still only consist of approaches providing limited symptom management. Our current efforts are advancing a novel biologically based treatment for the source of the debilitating symptomology of PD based on a tissue engineering approach that replaces lost dopaminergic neurons, directs axonal growth and connection to the striatum, and ultimately allows for specific, controlled external activation to regulate striatal dopamine levels. We anticipate that the continued development of dTELEs will serve as the foundation of a paradigm-shifting therapy for PD that will allow tailor-made treatments to address the root cause of PD motor symptoms rather than the symptoms themselves.

Acknowledgements (Optional):

References:
[1] F. Sun et al., “A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice,” Cell, vol. 174, no. 2, 2018, doi: 10.1016/j. cell.2018.06.042.
[2] G. DeMaagd and A. Philip, “Parkinson’s Disease and Its Management. Part 1: Disease Entity, Risk Factors, Pathophysiology, Clinical Presentation, and Diagnosis,”
[3] B. Thomas, “Parkinson’s Disease: From Molecular Pathways in Disease to Therapeutic Approaches,” Antioxidants and Redox Signaling, vol. 11, no. 9. 2009. doi: 10.1089/ars.2009.2697.
[4] Dayo O. Adewole et al., Development of Optically Controlled “Living Electrodes” With Long-Projecting Axon Tracts for A Synaptic Brain-Machine Interface. Sci. Adv. 7, eaay5347 (2021). DOI: 10.1126/sci adv.aay5347
[5] Struzyna, Laura A et al. “Tissue Engineered Nigrostriatal Pathway for Treatment of Parkinson's Disease.” Journal Of Tissue Engineering and Regenerative Medicine vol. 12,7 (2018): 1702-1716. doi:10.1002/term.2698