Picosecond Pulsed Electric Field-Induced Disaggregation of Polyglutamine Aggregates in Huntington’s Disease Neural Stem Cells

Picosecond Pulsed Electric Field-Induced Disaggregation of Polyglutamine Aggregates in Huntington’s Disease Neural Stem Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Picosecond Pulsed Electric Field-Induced Disaggregation of Polyglutamine Aggregates in Huntington’s Disease Neural Stem Cells Mackenzie Tardif-Kunk, Martina Zamponi, Emily Old, Augustine U. Anthony, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6486690/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Huntington’s disease (HD) is a neurodegenerative disorder marked by polyglutamine (PolyQ) aggregation and mitochondrial dysfunction, yet non-invasive methods to modulate these intracellular stressors remain limited. Here, we applied picosecond pulsed electric fields (psPEF)—an ultrashort bioelectronic modality—to patient-derived HD neural stem cells (NSCs) to assess changes in pathogenic protein aggregation, gene expression, and mitochondrial membrane potential. Using a custom 3D bioprinter-based stimulation platform, cells were exposed to non-contact electric fields of 20 or 40 kV/cm with subnanosecond pulse width (660 ps). Quantitative imaging and automated analysis revealed a significant reduction in aggregate size and aggresome burden within 30 minutes post-treatment, effects that persisted at 24 hours without compromising viability. HTT mRNA levels remained unchanged, supporting a post-translational mechanism of aggregate modulation. We also observed a transient redistribution of aggregates into the nuclear compartment and a field-dependent trend toward increased mitochondrial polarization, suggestive of broader proteostatic or bioenergetic effects. Transcript analysis revealed downregulation of PAX6 and CACNA1C, further implicating psPEF in modulating intracellular stress pathways. These findings represent the first evidence that ultrashort electric fields can reduce mutant HTT aggregation in a human HD model without genetic manipulation or membrane poration. Our results establish HD-NSCs as a scalable, disease-relevant platform for evaluating psPEF in neurodegenerative disease and support further exploration of dielectric mechanisms for intracellular remodeling. Collectively, this work introduces a contactless, non-invasive strategy for modulating protein aggregation and mitochondrial stress in human neural cells, offering a new direction for therapeutic development in proteopathy-driven conditions. picosecond pulse electric fields (psPEF) protein aggregation Huntington’s disease bioelectric modulation neural stem cells (NSCs) mitochondrial membrane potential (ΔΨm) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats within exon 1 of the Huntingtin ( HTT ) gene [1,2]. When this repeat exceeds approximately 35 units, the translated huntingtin protein (HTT) contains a pathogenic polyglutamine (PolyQ) tract that disrupts its native conformation, promoting the formation of β-sheet-rich amyloid fibrils. These protein aggregates accumulate in both the cytoplasm and nucleus, interfering with essential cellular processes such as transcription, proteostasis, and axonal transport [3–5]. In HD, aggregate toxicity disproportionately affects medium spiny neurons (MSNs) of the striatum, which are critical for motor coordination and cognitive function [6,4]. The progressive degeneration of MSNs leads to the hallmark triad of HD symptoms: involuntary movements, cognitive decline, and psychiatric disturbances. Despite improved understanding of HD pathophysiology, there remains no approved treatment that modifies disease progression or reduces aggregate burden [7]. Protein aggregation is increasingly recognized as a convergent pathological feature across neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s disease [8–14]. Therapeutic efforts to target aggregated proteins have traditionally relied on small molecules, antibodies, or gene-silencing approaches [15–26]. However, these methods often suffer from limited bioavailability, poor blood–brain barrier penetration, off-target effects, or suboptimal clearance of established aggregates. Alternative physical modalities that non-invasively modulate protein conformation or cellular pathways represent a promising but underexplored strategy. Pulsed electric fields (PEFs) have been widely studied for their ability to modulate cellular behavior through controlled electrical stimulation [27,28]. Conventional applications of PEFs in the microsecond to nanosecond range include reversible electroporation for gene delivery, irreversible electroporation for tissue ablation, and nanosecond PEFs (nsPEFs) for subcellular manipulation and cell death induction [29–35]. The biological effects of these pulses are heavily dependent on pulse duration, intensity, rise time, and the number of delivered pulses [36–43]. Among these, picosecond pulsed electric fields (psPEF) are the least characterized yet potentially the most selective modality for intracellular targeting. PsPEFs are defined by ultra-short pulse durations (< 1 ns) with subnanosecond rise times, which allow them to traverse the cell membrane without inducing membrane permeabilization due to their operation below the plasma membrane charging time constant (~ 75 ns) [44–46,31,30]. This property enables direct coupling of psPEFs to cytosolic components while minimizing collateral membrane damage. Emerging theoretical and experimental evidence suggests that psPEFs can act as a biophysical modulator of protein structure [47,48,31]. Molecular dynamics simulations have demonstrated that intense picosecond electric fields can induce a conformational shift in amyloidogenic proteins, such as β-amyloid and α-synuclein, favoring transitions from β-sheet to α-helical structures [49,50]. This mechanism has been proposed to underlie electric field-mediated disaggregation of protein fibrils. Experimental studies using purified protein models support this hypothesis, showing that localized electric fields can fragment amyloid plaques or prevent fibrillization [51,52]. However, no study to date has investigated psPEF effects on protein aggregates within a human disease-relevant neural cell context. This constitutes a critical gap in the translation of psPEF technology from a physical or biochemical phenomenon to a potential therapeutic tool. To address this, we applied psPEF to human neural stem cells derived from an HD patient to evaluate whether this non-invasive modality can modulate disease-relevant phenotypes, including polyglutamine aggregation and mitochondrial dysfunction. We utilized neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) reprogrammed from HD patient fibroblasts. These cells endogenously express full-length mutant huntingtin under native regulatory control and recapitulate early molecular features of HD, including PolyQ aggregation, altered calcium homeostasis, and mitochondrial dysfunction [53–58]. While mature neurons represent the ultimate target of neurodegenerative pathology, NSCs provide a scalable, tractable, and physiologically relevant model system for initial mechanistic studies. Their rapid expansion, consistent phenotype, and preservation of disease-relevant stress pathways make them ideally suited for high-content analysis of psPEF-induced biophysical and molecular responses. Moreover, early-stage investigation in NSCs enables controlled, within-line comparisons to quantify psPEF-mediated changes in aggregate burden, gene expression, and mitochondrial membrane potential (ΔΨm) without confounding variation from heterogeneous differentiation states. This approach establishes a foundational framework for future studies in differentiated neurons and more complex multicellular models. To ensure experimental tractability while capturing both immediate and intermediate responses to psPEF, we focused our analysis on 30-minute and 24-hour post-treatment time points. This timeframe was strategically chosen to detect acute changes in aggregate morphology, transcriptional modulation, and mitochondrial dynamics while avoiding variability introduced by extended in vitro culture. Although longer-term effects were not assessed in this study, these intervals provide critical insight into the initial cellular response to psPEF stimulation and form the basis for future investigations into the persistence and downstream impact of psPEF-mediated modulation. Field strengths of 20 and 40 kV/cm were selected based on previous bioelectric studies demonstrating intracellular modulation without overt membrane damage at these intensities [30,59,38,60]. These values represent physiologically relevant upper and mid-range intensities capable of modulating intracellular targets without inducing cellular toxicity. To deliver psPEF with spatial and temporal precision, we employed a custom-engineered 3D bioprinter platform equipped with a coaxial electrode probe connected to a picosecond pulse generator [61,59]. Using computational modeling and precise motion control, we exposed HD-NSCs to 1.8k pulses at electric field strengths of 20 kV/cm and 40 kV/cm. We then assessed mHTT aggregation, gene expression, and mitochondrial function at multiple time points post-exposure. This work represents the first in vitro demonstration of psPEF-mediated disaggregation of pathological protein aggregates in a human neural stem cell model of HD and provides foundational evidence for the use of psPEF as a non-invasive bioelectronic approach to proteopathy modulation. Materials and Methods Cell Lines and Culture The neuronal stem cell (NSC) lines used in this study were established in our laboratory from human HD patient-derived fibroblasts (GM04022; Coriell Institute) that had previously been reprogrammed to induced pluripotent stem cells (iPSCs) and have been phenotypically validated in earlier work [62,63]. HD-NSCs were cultured in STEMdiff™ Neural Progenitor Medium (Stemcell Technologies) supplemented with 1% antibiotic-antimycotic solution (Life Technologies) and maintained at 37°C in a humidified 5.0% CO₂ atmosphere. Media were replaced every 48 to 72 hours to ensure optimal nutrient availability and minimize metabolic stress. Cells were seeded onto Geltrex™-coated 6-well plates (1:100 dilution, Thermo Fisher Scientific) and passaged using TrypLE Express (Gibco) following the manufacturer’s protocol. All experiments were performed using early-passage cultures to minimize phenotype drift. For psPEF exposure and imaging-based analyses, a center-seeding method was used to standardize the exposure area. Geltrex™ (50–75 µL per well) was applied only to the center of each culture surface and incubated for 1 hour. Cells were then seeded directly onto this coated region at densities described per assay, followed by the addition of full medium volume after 1 hour. This technique is referred to herein as “center-seeding.” Characterization of iPSC-Derived Neural Stem Cells To confirm NSC identity and maintenance of neural progenitor characteristics, immunocytochemistry (ICC) was performed on adherent cultures. Cells were seeded at 3.5×10⁴ per well in Geltrex™-coated 8-well chamber slides and fixed after 48 hours using 10% neutral-buffered formalin. Permeabilization was carried out using 0.1% NP-40 in PBS for 10 minutes, followed by blocking with 10% normal goat serum for 1 hour. Primary antibodies targeting canonical NSC markers were applied in PBS containing 1% goat serum for 1 hour at room temperature. These included SOX1 (ab87775, 1:200, Abcam), SOX2 (ab97959, 1:1000, Abcam), and PAX6 (13B10-1A10, 1:200, Thermo Fisher Scientific) to confirm neuroectodermal lineage commitment, and nestin (MA1-110, 1:100, Thermo Fisher Scientific) to assess intermediate filament expression. After three PBS washes, cells were incubated with Alexa Fluor-conjugated secondary antibodies (488 and 568; 1:1000, Thermo Fisher) for 1 hour. DAPI (1:1000) was used to counterstain nuclei. Slides were mounted with Fluoromount-G® and imaged using a Zeiss Axio Observer Z1 inverted microscope under standardized acquisition settings. Mutant HTT Fragment Analysis To confirm the presence of expanded CAG repeats in HTT , triplet-primed PCR (TP-PCR) followed by capillary electrophoresis was performed using previously validated protocols [64,62]. Fluorescently labeled primers targeting the HTT locus were used, and PCR amplification was carried out with Platinum™ Taq High Fidelity polymerase under optimized conditions. Amplicons were denatured and separated using a 3130 Genetic Analyzer (Applied Biosystems). Size distributions were analyzed with GeneMarker® v2.6.7 using custom bin definitions created from Coriell reference HD cell lines. psPEF Delivery Using 3D Printer-Based Electrode System Picosecond pulsed electric fields (psPEF) were delivered using a custom-designed in vitro stimulation platform that integrates a high-speed pulse generator with a programmable 3D motion control system. The exposure system was based on a modified FELIX 3.0 3D printer (FELIXrobotics, Ijsselstein, Netherlands) equipped with a coaxial electrode probe mounted to the print head, enabling spatially controlled non-contact stimulation of cell monolayers [59]. The electrode probe consisted of parallel tungsten wires (0.1 mm diameter), each measuring approximately 1.5 cm in length. The wires were soldered to the signal and ground terminals of a 50 Ω coaxial transmission line and embedded in epoxy insulation, leaving 1.0 cm of exposed tungsten at the tip. Electrodes were positioned 1 mm apart (edge-to-edge), forming a narrow, high-field fringing configuration. The probe was mounted to the X-axis carriage and vertically calibrated using the mechanical pitch of the printer’s leadscrew to maintain a consistent 200 µm (0.2 mm) standoff from the culture surface. High-voltage pulse generation was provided by an FPG 10-10PM1 pulse generator (FID GmbH, Burbach, Germany), capable of producing unipolar Gaussian pulses with amplitudes up to 10 kV into a 50 Ω load. For this study, the pulse amplitude was adjusted to 2.0 kV and 4.0 kV, corresponding to calculated electric field strengths of 20 kV/cm and 40 kV/cm at the cell surface. Each pulse exhibited a rise time of 250–350 ps and a full-width at half-maximum (FWHM) duration of 660 ps, depending on the voltage setting. Pulses were delivered at a fixed repetition rate of 1 kHz, consistent with the generator’s internal triggering configuration. The pulse repetition frequency of 1 kHz was chosen based on common practice within ultrashort pulse bioelectric studies to balance effective stimulation with minimal thermal accumulation [39,65]. Prior studies employing similar ultrashort pulses at frequencies around 1 kHz have demonstrated effective intracellular modulation without observable thermal or cytotoxic effects. Field strength calibration was verified through direct voltage measurements and confirmed using FDTD simulations in CST Microwave Studio. Cells were exposed to a total of approximately 1,800 pulses as the probe traversed an S-shaped scan path generated using custom MATLAB scripts converted to G-code. The spatial pulse delivery ensured uniform treatment across the designated cell zone without overlapping tracks. Prior to pulse delivery, culture medium was removed and replaced with PBS to reduce dielectric damping and avoid field dispersion caused by high ionic conductivity. This 3D bioprinter-based psPEF delivery system allows for highly reproducible, spatially uniform stimulation under tightly controlled electrical and mechanical parameters, enabling novel investigations of subcellular responses to non-contact ultrashort electric fields. All analyses were performed at 30 minutes and 24 hours post-exposure, selected to capture both acute and intermediate cellular responses to psPEF while minimizing variability associated with prolonged in vitro culture. Cell Viability Assessment Cell viability following psPEF exposure was assessed using the LIVE/DEAD™ Cell Imaging Kit (Thermo Fisher Scientific), which distinguishes viable cells via intracellular esterase activity (calcein AM, green fluorescence) and dead cells by membrane-impermeable BOBO-3™ iodide (red fluorescence). HD-NSCs were center-seeded at 3×10⁴ cells per well in Geltrex™-coated 12-well plates and incubated for 48 hours before psPEF exposure at 0, 20, or 40 kV/cm. After pulsing, cells were allowed to recover in their respective culture media for 30 minutes. Following recovery, the LIVE/DEAD™ reagents were added directly to the culture medium at a 1:1 dilution and incubated at room temperature for 15 minutes in the dark. Cells were then washed with PBS to remove excess dye and returned to dye-free media. Fluorescent images were captured using a Zeiss Axio Observer Z1 inverted microscope with consistent settings across all conditions. A total of twelve images per treatment group (three per well from four replicates) were collected from non-overlapping fields of view. Viable and non-viable cells were manually counted by a blinded observer using ImageJ. The percentage of live cells per field was calculated and averaged across replicates. Group differences were analyzed as described in the Statistical Analysis section. This method allowed for the assessment of cytotoxic effects of psPEF under the tested conditions while minimizing observer bias and ensuring standardized acquisition. Immunocytochemistry and Confocal Imaging For aggregate quantification, human HD neural stem cells (NSCs) were seeded at 2 × 10⁴ cells per dish onto Geltrex™-coated glass-bottom dishes (MatTek; 35-mm dish, No. 1.5 coverslip, 10 mm glass diameter) using the center-seeding method. Cells were allowed to adhere for 48 hours prior to psPEF exposure. Following stimulation at 0, 20, or 40 kV/cm, cells were fixed at 30 minutes or 24 hours post-treatment using ice-cold 100% methanol at − 20°C for 10 minutes. Fixed cells were blocked with 10% normal goat serum in PBS for 1 hour and stained with primary antibodies against polyglutamine-expanded proteins (PolyQ; Sigma MAB1574, 1:1000) and huntingtin (HTT; Abcam ab109115, 1:1000), diluted in 1% goat serum in PBS for 1 hour at room temperature. Secondary antibodies conjugated to Alexa Fluor 488 and 568 (Thermo Fisher, 1:1000) were used for detection. DAPI was used for nuclear counterstaining. Samples were mounted in Fluoromount-G® and sealed for imaging. Confocal imaging was performed on a Leica Stellaris 5 system equipped with a DMI8 inverted microscope, HyD detectors, and a tunable white light laser. Imaging was conducted using a 40x oil immersion objective (NA 1.40). Acquisition settings were rigorously standardized across all experimental conditions, including a 1024 × 1024-pixel resolution, a z-step size of 0.3 µm, pinhole set to 1 Airy unit, and laser power, detector gain, and offset fixed across all sessions after optimization on control samples. To reduce noise and enhance signal fidelity, line averaging (3–4×) and frame accumulation (2×) were applied. At least ten non-overlapping fields of view were imaged per condition, each containing 10–20 cells. All imaging was performed in a blinded and randomized fashion. Aggregate Quantification Quantitative image analysis of PolyQ aggregates was performed using Fiji (ImageJ v1.53) and the AggreCount macro was specifically chosen due to its automated, objective, and reproducible quantification of aggregates [66]. This approach eliminated subjective interpretation and minimized potential observer bias, ensuring robust, reproducible data irrespective of operator experience. Prior to analysis, raw confocal z-stacks were converted to 16-bit grayscale and projected into two-dimensional images using the “sum slices” function to preserve intensity values across all z-planes. This approach ensured uniform image input and was well-suited to detecting small, high-intensity puncta consistent with protein aggregates. The AggreCount macro was run in segmentation-based “cell processing” mode using validated parameters. Thresholding was manually calibrated using non-pulsed control samples to ensure consistent background exclusion across all groups. Aggregates were segmented using a minimum area threshold of 0.2 µm² and a maximum of 20 µm². Aggregates exceeding 4.0 µm² were classified as “aggresomes.” Perinuclear localization was defined as aggregate proximity within 10 pixels of the nuclear boundary. The plugin was configured to exclude nuclei smaller than 50 µm² and cells smaller than 75 µm²; both cell and nuclear segmentation used a strictness value of 5 to ensure edge fidelity and minimize overlap artifacts. Quantitative outputs included aggregate count, mean aggregate size, total aggregate area, aggresome count, and compartment-specific distribution (cytosolic, perinuclear, nuclear) normalized per nucleus. All analyses were conducted using batch processing to maintain parameter consistency. Fluorescence localization was verified using orthogonal slice views and maximum intensity projections to confirm the spatial accuracy of aggregate classification. Comparisons between treatment groups and localization categories were conducted as outlined in the Statistical Analysis section. Quantitative RT-PCR HD-NSCs were center-seeded on Geltrex™ in 12-well plates at a density of 4x104 cells. Cells were pulsed with 4 replicates at 20 kV/cm or 40 kV/cm or designated as a non-pulsed control. Total RNA was isolated 24 hours after pulsing using TRIzol (Invitrogen) according to the manufacturer’s protocol. RNA quantity and quality were determined by the absorbance at 260/280 nm using a NanoDrop 2000 (Thermo Fisher). Any genomic DNA was removed from the RNA sample by deoxyribonuclease I (Invitrogen) and the manufacturer’s protocol. The High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used to synthesize cDNA from the RNA samples (1 mg). Quantitative RT-PCR was performed on a QuantStudioTM 7 Flex Real-Time PCR System (Applied Biosystems). TaqMan Gene Expression Assays (Applied Biosystems) were used to quantify mRNA for the following genes: HTT (Hs00918174_m1), CACNA1C (Hs00167681_m1), and PAX6 (Hs00240871_m1). ACTB (Hs01060665_g1) served as the endogenous housekeeping gene. All samples were analyzed in triplicate using TaqMan Fast Advanced Master Mix (Life Technologies) and 5 ng of cDNA per reaction according to the manufacturer’s protocol. The 2 −ΔΔCt method was used to calculate the fold-changes relative to the no-pulse control (NPC) [67]. Group differences in gene expression were evaluated as described in the Statistical Analysis section. TMRE Imaging and Analysis HD-NSCs were center-seeded onto Geltrex™-coated 35 mm glass-bottom dishes (MatTek; No. 1.5 coverslip, 10-mm glass area) at a density of 2×10⁴ cells per dish and incubated for 48 hours prior to psPEF exposure. After pulsing at 0, 20, or 40 kV/cm, cells were immediately incubated in pre-warmed media containing 50 nM tetramethylrhodamine ethyl ester (TMRE; Thermo Fisher Scientific), a cell-permeant, cationic dye that accumulates in polarized mitochondria. Following 30 minutes of incubation under standard conditions (37°C, 5% CO₂), cells were washed twice with PBS and returned to fresh medium without TMRE prior to imaging. Live imaging was performed every 5 minutes for 30 minutes using a Leica Stellaris 5 confocal microscope with a DMI8 inverted platform, HyD detectors, and a 40x oil immersion objective (NA 1.40). TMRE was excited at 549 nm, and emission was collected at 574 nm. Acquisition parameters—including laser power, gain, detector settings, and pinhole diameter—were held constant across all samples and time points. Z-stacks were collected with identical slice numbers and spacing, allowing consistent volumetric fluorescence comparison across conditions. Image analysis was conducted using a custom macro in Fiji (ImageJ v1.53). Each z-stack image was converted to 8-bit grayscale and thresholded using Otsu’s method to isolate TMRE-positive signal. [68] A binary mask was applied, and integrated fluorescence intensity was quantified within the thresholded region using the "Analyze Particles" function. No manual ROI selection, background subtraction, or filtering was applied to preserve consistency. Each condition was represented by at least six replicate regions of interest (ROIs), each containing approximately 10–20 cells. The same fields of view were tracked over the 30-minute time course. Raw values were used for statistical comparison. Time- and treatment-related effects were analyzed using the approach described in the Statistical Analysis section. Statistical Analysis All statistical analyses were performed using GraphPad Prism v10.4.1. For all parametric comparisons involving three or more groups, one-way analysis of variance (ANOVA) was used followed by Tukey’s post hoc test to evaluate pairwise differences. This approach was applied to quantitative RT-PCR data, viability assays, and aggregate burden metrics across treatment conditions. For comparisons involving categorical variables such as subcellular aggregate localization, chi-squared analysis was employed to assess distributional shifts across compartments. Time-course data from TMRE imaging were analyzed using a two-way repeated measures ANOVA to evaluate the effects of time and treatment on mitochondrial membrane potential. Assumptions of normality and sphericity were assessed using the Shapiro-Wilk and Mauchly’s tests, respectively. In instances where assumptions were violated, nonparametric alternatives including the Friedman test and aligned rank transform were applied. Statistical significance was defined as p < 0.05. Where statistical thresholds were not met, biologically relevant trends were reported when supported by effect size, directionality, and consistency across replicates. Data are presented as mean ± standard deviation unless otherwise noted. Results Characterization of Neural Stem Cells by Immunocytochemistry and Genotyping To verify the identity and regional specification of the iPSC-derived HD neural stem cells (NSCs), immunocytochemistry was performed to assess expression of key neurodevelopmental markers (Fig. 1 A). SOX1 and SOX2, transcription factors associated with neuroectodermal lineage commitment and maintenance of NSC identity, exhibited strong nuclear localization in the majority of cells. PAX6, a regulator of forebrain patterning, was similarly localized to the nucleus in a broad subset of cells, consistent with successful differentiation toward a dorsal forebrain lineage. Nestin, a marker of intermediate filaments in proliferating NSCs, displayed filamentous cytoplasmic staining throughout the culture. These patterns confirm a neuroepithelial-like NSC phenotype, supportive of subsequent differentiation into central nervous system lineages. [Insert Fig. 1 here] To confirm our previous characterization of the HD genotype of the NSC line (GM04022), triplet-primed PCR (TP-PCR) followed by capillary electrophoresis was performed (Fig. 1 B) [63]. Fragment analysis revealed heterozygous CAG repeat lengths of 18 and 44, consistent with a pathogenic HD allele and a normal allele. These results verify that the cell line retains the genetic hallmark of Huntington’s disease and is suitable for modeling polyglutamine pathology in vitro. psPEF Exposure Does Not Compromise Cell Viability Given that PEFs can induce membrane permeabilization or cell death depending on pulse parameters, we evaluated the cytotoxic potential of psPEF exposure using a LIVE/DEAD™ viability assay. HD-NSCs were exposed to 20 or 40 kV/cm psPEF and assessed 30 minutes post-exposure (Fig. 2 A). Live cells were defined by intracellular esterase activity (calcein AM), while non-viable cells were labeled by membrane-impermeant BOBO-3™ iodide. No statistically significant differences in viability were detected at either field strength when compared to non-pulsed controls (20 kV/cm: p = 0.536; 40 kV/cm: p = 0.475), indicating that psPEF treatment under these conditions does not compromise membrane integrity or induce acute cytotoxicity (Fig. 2 B)​. [Insert Fig. 2 here] psPEF Reduces mHTT Aggregate Burden in HD-NSCs To determine whether psPEF could influence protein aggregation, HD-NSCs were immunoassayed for mutant huntingtin (PolyQ) following exposure to 0, 20, or 40 kV/cm psPEF. Compared to untreated controls, psPEF-treated cells exhibited visibly reduced PolyQ puncta intensity and size at both 30 minutes and 24 hours post-exposure (Fig. 3 A). Confocal image stacks were analyzed using the AggreCount macro to quantify total PolyQ-positive puncta, average aggregate size, and aggresome burden. [Insert Fig. 3 here] Interestingly, despite significant reductions in aggregate size and aggresome load, the total number of aggregates per cell did not significantly decrease at either field strength (Fig. 3 B). This observation may suggest a remodeling or fragmentation of larger aggregates into smaller subunits rather than complete clearance. Thus, the psPEF-induced disaggregation may primarily reduce aggregate pathogenicity by decreasing their size and altering intracellular distribution, rather than eliminating aggregate formation entirely. However, the average aggregate area per cell decreased by 66% ( p = 0.0174) and 63% ( p = 0.0224) for 20 and 40 kV/cm, respectively, 30 minutes after pulsing (Fig. 3 C). These reductions were further amplified at 24 hours, with aggregate areas reduced by 74% ( p = 0.0049) and 77% ( p = 0.0052) for 20 and 40 kV/cm, respectively. Similarly, average individual aggregate size decreased (Fig. 3 D) by 58–69% across both field strengths and time points (all p = 0.0193, 0.0092, 0.0196, 0.0058, respectively). The number of aggresomes per cell (≥ 4.0 µm²) was also significantly reduced (Fig. 3 E)—by up to 56% at 30 minutes and 43% at 24 hours ( p < 0.0001, p = 0.0004, p = 0.0076, respectively). Aggregate segmentation parameters were calibrated using non-pulsed controls and visually validated against raw image stacks to confirm detection fidelity across subcellular compartments. This ensured consistent identification of small puncta and excluded background artifacts. Subcellular Distribution of PolyQ Aggregates Is Differentially Modulated by psPEF Following psPEF exposure, compartment-specific changes in aggregate burden were observed across cytosolic, perinuclear, and nuclear regions. In the cytosolic compartment (Fig. 4 A & 4 B), both aggregate number and total area were significantly reduced at 30 minutes post-treatment. Aggregate counts declined by 59–62% ( p < 0.0001), and total aggregate area decreased by 79–81% ( p = 0.0012, 0.0007, respectively) at both 20 and 40 kV/cm. These reductions persisted through 24 hours post-exposure, indicating a durable effect of psPEF on cytosolic aggregate load. [Insert Fig. 4 here] In the perinuclear compartment (Fig. 4 C–D), psPEF exposure did not significantly alter aggregate number or area at 30 minutes post-treatment. However, a delayed 33% reduction in aggregate count was observed at 24 hours following 20 kV/cm stimulation ( p < 0.01), indicating a time-dependent effect on perinuclear aggregate clearance (Fig. 4 C). Notably, the total area of perinuclear aggregates per cell decreased by 62–67% at 30 minutes ( p = 0.0139, 0.0217, respectively) and by 73–74% at 24 hours ( p = 0.0036, 0.0072, respectively), consistent with a sustained morphological remodeling of perinuclear aggregate burden (Fig. 4 D). In the nuclear compartment (Fig. 4 E & 4 F), psPEF exposure led to a transient increase in aggregate number at 30 minutes (20 kV/cm: 42.6%, 40 kV/cm: 44.0%; p < 0.0001), without a significant change in total aggregate area. This transient nuclear accumulation coincided with reductions in cytosolic and perinuclear aggregates. The overall distribution of aggregates across compartments is summarized in Fig. 4 G. In untreated cells, aggregates were predominantly cytosolic (45.2%), followed by perinuclear (40.0%) and nuclear (14.9%) localization. psPEF exposure shifted this balance toward the nucleus at 30 minutes, particularly at higher field strengths. This redistribution was partially reversed by 24 hours, suggesting a dynamic reorganization of aggregate localization rather than degradation alone. Pulse intensity did not significantly alter the direction of redistribution. Gene Expression Changes Following psPEF Treatment Quantitative RT-PCR was performed 24 hours after psPEF exposure to evaluate whether observed changes in aggregate burden could be attributed to transcriptional regulation. No significant differences in HTT mRNA expression were observed ( p = 0.479 for 20 kV/cm; p = 0.622 for 40 kV/cm). However, both PAX6 and CACNA1C transcripts were significantly downregulated following exposure, with more pronounced suppression at higher field strength. PAX6 levels declined by ~ 26% ( p = 0.0195) at 40 kV/cm, while CACNA1C expression decreased by ~ 23% ( p = 0.0493), suggesting that psPEF modulates the expression of neurodevelopmental and ion channel genes independently of HTT transcription​ (Fig. 5 ). [Insert Fig. 5 here] psPEF May Influence Mitochondrial Polarization Given that mitochondrial dysfunction and reduced membrane potential are hallmark features of Huntington’s disease pathology, we assessed whether psPEF stimulation influences mitochondrial polarization in HD-NSCs. [69–73] Cells were loaded with TMRE, a potentiometric dye that accumulates in mitochondria proportional to membrane potential (ΔΨm), and live-cell confocal imaging was performed over a 30-minute time course following psPEF exposure. [Insert Fig. 6 here] Qualitative assessment of TMRE fluorescence revealed modest increases in signal intensity in psPEF-treated cells, particularly in the 40 kV/cm group, relative to non-pulsed controls (Fig. 6 A). Fluorescence enhancement appeared most pronounced at 5- and 30-minutes post-treatment, suggesting a delayed but potentially sustained bioenergetic response. These differences were visualized using a fire lookup table (LUT) applied post-quantification to highlight regional variations in signal. Quantitative analysis of integrated TMRE intensity showed a trend toward elevated membrane potential in psPEF-treated groups compared to control, with the most consistent increase observed in the 40 kV/cm condition (Fig. 6 B). Although differences did not reach statistical significance in two-way repeated measures ANOVA, the directionality of the response—especially given the known baseline mitochondrial depolarization in HD cells—suggests that psPEF may influence mitochondrial function in a non-lethal and potentially restorative manner. These findings support further exploration of psPEF as a modulator of mitochondrial health in models of proteopathic stress. Discussion The application of psPEF in biomedical research represents a novel frontier within bioelectrics, uniquely suited to modulate intracellular processes without irreversibly disrupting membrane integrity [31,30,60]. Unlike microsecond and nanosecond pulses—commonly employed for electroporation, apoptosis induction, or electrochemotherapy—psPEFs operate at subnanosecond timescales, allowing interaction with intracellular targets prior to plasma membrane charging. This study provides the first evidence that psPEF can non-lethally reduce mHTT aggregation and modulate mitochondrial polarization in a human cellular model of Huntington’s disease, supporting its potential as a bioelectronic approach to modulating HD-relevant intracellular stress. These findings advance the concept of psPEF as a bioelectronic tool for modulating proteostasis in neurodegenerative disease models. Our data demonstrate that single-exposure psPEF treatment at 20 or 40 kV/cm reduces both the size and area of mHTT aggregates in HD-NSCs. This reduction was maintained at 24 hours post-treatment, suggesting durable reorganization or clearance of aggregate material. Importantly, the total number of aggregates per cell was largely unchanged, supporting a model in which psPEF primarily alters aggregate morphology or compactness rather than affecting seeding or transcription. This aligns with modeling studies suggesting that ultrashort electric fields can induce β-sheet to α-helix transitions in amyloidogenic proteins, destabilizing aggregate structures [74]. Supporting this mechanism, HTT transcript levels remained unchanged following psPEF exposure, indicating that the disaggregation effect was not attributable to reduced expression. Instead, the effects appear to occur at the post-translational level. The dose-responsiveness of this effect—where more robust changes occurred at 40 kV/cm—further supports a field-strength dependent biophysical modulation of protein structure or intracellular trafficking. It remains to be determined whether the observed reduction in aggregate burden reflects true disaggregation, intracellular redistribution, or degradation via proteasomal or autophagic clearance. Future studies incorporating proteostasis pathway reporters and selective inhibitors will be critical to resolve these possibilities. Interestingly, the spatial distribution of aggregates was differentially affected by psPEF. Cytosolic aggregates exhibited the most pronounced reductions, whereas nuclear aggregates transiently increased at 30 minutes before returning to baseline. One hypothesis is that larger aggregates may fragment and redistribute into nuclear compartments where clearance is slower or more compartmentalized. Alternatively, psPEF may differentially affect nuclear envelope permeability or aggregate trafficking mechanisms. While speculative, these results underscore the importance of subcellular localization in determining psPEF responses. Our primary aim was to assess the modulation of existing aggregate burden within HD-derived cells. This within-line design enables interpretation of treatment effects without confounding by interline variability. Future studies incorporating isogenic WT-NSCs or time-course recovery analysis could further validate the specificity and longevity of the observed disaggregation effects. While neurons are the ultimate target of HD pathology, NSCs offer a biologically relevant and experimentally tractable model system for probing early aggregation phenotypes [54–57]. Their preserved stress response pathways, rapid expansion, and lineage consistency make them well suited for high-content, mechanistically focused investigations of intracellular remodeling [75]. Prior studies have used NSCs to model protein aggregation dynamics and validate modulatory interventions before translation to mature neuronal systems [53,57,58]. In addition to structural changes, we observed downregulation of two genes, PAX6 and CACNA1C , following psPEF treatment. While these targets were not initially hypothesized, their modulation raises interesting possibilities. PAX6 is a neurodevelopmental transcription factor associated with progenitor maintenance and identity, and its reduction may suggest a shift toward neuronal maturation or stress adaptation [76]. CACNA1C , encoding the Cav1.2 L-type calcium channel, regulates calcium influx and synaptic plasticity and is dysregulated in several neuropsychiatric conditions. Suppression of CACNA1C could reflect altered excitability or intracellular calcium homeostasis—both processes influenced in HD pathology [77,17]. The precise relationship between psPEF exposure, transcriptional regulation, and protein aggregate dynamics remains to be fully elucidated. We also observed a trend toward increased ΔΨm as assessed by TMRE staining, most notably at the higher field strength —which is intriguing given that HD cells typically start from a chronically depolarized baseline [69–71]. While these results did not reach statistical significance, possibly due to sample variability and temporal resolution limitations, the directionality aligns with reports that aggregate reduction may restore mitochondrial polarization in HD models [78]. Two, not mutually exclusive, mechanisms could make such a transient hyperpolarization feasible. First, modelling studies predict that each sub‑nanosecond pulse generates opposite charge accumulations across the inner mitochondrial membrane, producing momentary anode‑facing hyperpolarization before the net depolarizing phase dominates; integrating many pulses at 1 kHz could therefore bias the average ΔΨm upward in the short term [47,30,79]. Second, nsPEF exposure at sub‑lethal doses has been shown to stimulate electron‑transport flux and reduce proton leak, thereby increasing ΔΨm in other cell types—a hormetic response later confirmed in HD neurons when PINK1 activation restored ΔΨm and viability [80–83]. Live-cell potentiometric assays like TMRE are inherently variable due to mitochondrial network heterogeneity and cell-to-cell differences in dye loading. The modest sample size and early time-point focus may have limited statistical power despite biologically plausible trends. The 30-minute window was chosen to capture acute, primary responses in mitochondrial potential that may reflect direct effects of psPEF. Although post-hoc TMRE staining at 24 hours could theoretically provide insight into longer-term adaptations, we elected not to perform this due to both technical and biological limitations. Delayed TMRE application introduces greater susceptibility to dye-loading artifacts and reduced dynamic range, particularly in cells with altered metabolic states. Moreover, mitochondrial remodeling over extended intervals in HD models can obscure primary psPEF effects, making early time points more reliable for mechanistic interpretation. Future use of genetically encoded voltage indicators may allow improved resolution of these dynamics. Mechanistically, one potential pathway linking psPEF exposure to these effects involves intracellular calcium signaling. Several studies have reported that subnanosecond electric fields can induce calcium influx through voltage-gated calcium channels (VGCCs), particularly in cells that endogenously express these channels [74]. In our model, VGCC-mediated activation is plausible and consistent with prior findings that psPEF induces calcium transients without gross electroporation. However, we acknowledge that VGCC gating typically operates on microsecond timescales, which far exceeds the duration of our stimulus. Thus, conventional depolarization is unlikely to fully explain the observed effects. Instead, we propose that dielectric mechanisms—such as local field-induced protein conformational changes or water dipole reorientation—may contribute to transient activation or sensitization of these channels [37,84]. In parallel with potential ion channel effects, we also propose that psPEF exposure may act directly on misfolded proteins or organelles via field-induced biophysical mechanisms. Given the subnanosecond rise time and field strengths applied here (20–40 kV/cm), psPEF may transiently disrupt stabilizing interactions within polyglutamine aggregates through dipolar polarization or electroconformational stress. This hypothesis is supported by molecular dynamics simulations showing that ultrashort electric fields can induce β-sheet to α-helix transitions in amyloidogenic proteins, destabilizing fibrillar structures without the need for chemical chaperones [74,49]. Additionally, psPEF may influence cytoskeletal transport or cytoplasmic viscosity, facilitating the redistribution of aggregates observed post-treatment. Though membrane nanoporation is unlikely under these conditions, picosecond pulses have been shown to affect organelle membrane potential and protein function through dielectric polarization and water dipole reorientation [84,60,85,61]. These complementary mechanisms may help explain the observed changes in aggregate morphology and mitochondrial behavior while maintaining cell viability. Alternatively, nanoporation cannot be entirely ruled out, particularly at higher field strengths. Although subnanosecond pulses are typically below the membrane charging time constant, molecular dynamics simulations suggest that extreme field strengths can induce water finger formation and transient nanopores within picoseconds under specific conditions [86]. It remains possible that some degree of non-lethal nanoporation or indirect ion channel modulation occurs, particularly in more exposed cytosolic regions. Therefore, we interpret VGCC activation as a likely but not exclusive pathway, and future studies with channel blockers, high-speed imaging, or electrophysiological recordings will be required to delineate these mechanisms more definitively. Further, while prior research using nsPEFs often emphasizes apoptosis induction or irreversible electroporation for cancer ablation, our study deliberately applies lower field strengths to avoid cytotoxicity. Indeed, we observe no loss in viability across conditions, supporting the concept that psPEF can modulate intracellular targets non-destructively. This distinction underscores the versatility of psPEF as a biophysical tool: unlike nsPEF-driven apoptosis or membrane rupture, psPEF may enable intracellular remodeling with minimal off-target damage when appropriately tuned [78,74]. While our study focused on early and intermediate responses, the 24-hour window was intentionally chosen to capture mechanistic changes while ensuring experimental consistency. Longer-term studies will be needed to determine whether psPEF-induced disaggregation leads to functional recovery, neuronal differentiation, or downstream clearance pathways. However, given the urgency of advancing psPEF as a novel modality, this window was appropriate for delineating short-term cellular responses. While no significant temperature rise was observed in earlier studies using similar conditions (e.g., via pH-sensitive dye methods), future iterations of the system will incorporate real-time thermal imaging or thermocouple feedback to confirm the absence of joule heating. Although simulation geometries were simplified and did not include the dielectric properties of well plate materials or fluid layers, ongoing refinements aim to improve the accuracy of modeled field distributions. Looking forward, the non-contact nature of psPEF presents a critical advantage for translational application. Unlike traditional electrode-based systems that require direct physical contact, future implementations could leverage dielectric-loaded wideband antennas to deliver focused picosecond pulses to targeted tissues at depth. Recent simulations and in situ measurements demonstrate that such antennas can transmit high-intensity fields (≥ 20 kV/cm) several centimeters into subcutaneous targets, including the brain, with spatial precision in the sub-centimeter range [61]. This proof-of-concept antenna design addresses key engineering challenges in minimizing reflection losses at tissue-air interfaces and maintaining pulse fidelity. When combined with the non-lethal, intracellularly-targeted effects demonstrated in our study, these advances support a broader vision for psPEF as a contactless therapeutic platform for modulating proteostasis in vivo. In summary, our findings demonstrate that picosecond pulsed electric fields can non-lethally remodel polyglutamine aggregates in human HD neural stem cells, inducing durable changes in aggregate morphology and localization without compromising cell viability. While the precise molecular pathways remain to be elucidated, our data suggest that psPEF exerts its effects through a combination of field-induced biophysical mechanisms and transcriptional modulation. The lack of HTT transcript change, coupled with morphological disaggregation and redistribution, points to post-translational processes as a key component of the response. Importantly, this study provides proof-of-concept that psPEF can be leveraged as a contactless modality to interrogate and modulate proteostasis within intact human cells. These findings lay the groundwork for future exploration of psPEF as a therapeutic or investigative tool across a broad spectrum of aggregation-driven neurodegenerative diseases. While the present study focused on morphological and molecular changes in aggregate burden, future studies will be needed to determine whether psPEF-induced disaggregation translates into functional recovery. Assessments of synaptic signaling, differentiation status, and calcium dynamics will be essential to establish therapeutic relevance in mature neuronal systems. Declarations Funding: This work was funded by institutional start-up support from the Ellmer College of Health Sciences at Old Dominion University. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author Contributions: M.T. performed the experiments, conducted data analysis, and contributed to manuscript preparation. M.Z., E.O., A.U.A., and C.K. assisted with experimental procedures. T.R.C. contributed to the statistical analysis of the data. R.A.P. provided technical expertise related to pulse delivery parameters. P.A.M., P.C.S., and R.D.B. jointly conceived and designed the study, supervised the research, interpreted the findings, and critically edited the manuscript. Data Availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Clinical Trial Number: Not applicable Material Transfer Agreement: The Huntington’s disease neural stem cell lines and associated imaging data can be provided by Old Dominion University pending scientific review and a completed material transfer agreement. Requests for these materials should be submitted to: Peter A. Mollica ( [email protected] ). References Gusella JF, MacDonald ME, Ambrose CM, Duyao MP (1993) Molecular genetics of Huntington's disease. Arch Neurol 50 (11):1157-1163 MacDonald ME, Ambrose CM, Duyao MP, Myers RH, Lin C, Srinidhi L, Barnes G, Taylor SA, James M, Groot N, MacFarlane H, Jenkins B, Anderson MA, Wexler NS, Gusella JF (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. 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Nature neuroscience 5 (8):731-736. doi:10.1038/nn884 Reddy PH, Shirendeb UP (2012) Mutant huntingtin, abnormal mitochondrial dynamics, defective axonal transport of mitochondria, and selective synaptic degeneration in Huntington's disease. Biochim Biophys Acta 1822 (2):101-110. doi:10.1016/j.bbadis.2011.10.016 Shirendeb UP, Calkins MJ, Manczak M, Anekonda V, Dufour B, McBride JL, Mao P, Reddy PH (2012) Mutant huntingtin's interaction with mitochondrial protein Drp1 impairs mitochondrial biogenesis and causes defective axonal transport and synaptic degeneration in Huntington's disease. Human molecular genetics 21 (2):406-420. doi:10.1093/hmg/ddr475 Lontay B, Kiss A, Virag L, Tar K (2020) How Do Post-Translational Modifications Influence the Pathomechanistic Landscape of Huntington's Disease? A Comprehensive Review. International journal of molecular sciences 21 (12). doi:10.3390/ijms21124282 Braun MM, Puglielli L (2022) Defective PTEN-induced kinase 1/Parkin mediated mitophagy and neurodegenerative diseases. Frontiers in cellular neuroscience 16:1031153. doi:10.3389/fncel.2022.1031153 Ibrahimi N, Vallet L, Andre FM, Rivaletto M, Novac BM, Mir LM, Pecastaing L (2023) An Overview of Subnanosecond Pulsed Electric Field Biological Effects: Toward Contactless Technologies for Cancer Treatment. Bioelectricity 5 (2):76-98. doi:10.1089/bioe.2022.0031 Carter RL, Chen Y, Kunkanjanawan T, Xu Y, Moran SP, Putkhao K, Yang J, Huang AH, Parnpai R, Chan AW (2014) Reversal of cellular phenotypes in neural cells derived from Huntington's disease monkey-induced pluripotent stem cells. 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Ieee T Plasma Sci 51 (7):1682-1717. doi:10.1109/Tps.2023.3281339 Qiu H, Xiao S, Joshi RP (2014) Simulations of Voltage Transients Across Intracellular Mitochondrial Membranes Due to Nanosecond Electrical Pulses. Ieee T Plasma Sci 42 (10):3113-3120. doi:10.1109/TPS.2014.2308871 Khalil B, El Fissi N, Aouane A, Cabirol-Pol MJ, Rival T, Lievens JC (2015) PINK1-induced mitophagy promotes neuroprotection in Huntington's disease. Cell death & disease 6 (1):e1617. doi:10.1038/cddis.2014.581 Hamamoto T, Ohno K, Kagawa Y (1982) Net adenosine triphosphate synthesis driven by an external electric field in rat liver mitochondria. Journal of biochemistry 91 (5):1759-1766. doi:10.1093/oxfordjournals.jbchem.a133868 Goswami I, Perry JB, Allen ME, Brown DA, von Spakovsky MR, Verbridge SS (2018) Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration. Biophysical journal 114 (12):2951-2964. doi:10.1016/j.bpj.2018.04.047 Asadipour K, Hani MB, Potter L, Ruedlinger BL, Lai N, Beebe SJ (2024) Nanosecond Pulsed Electric Fields (nsPEFs) Modulate Electron Transport in the Plasma Membrane and the Mitochondria. Bioelectrochemistry 155:108568. doi:10.1016/j.bioelechem.2023.108568 Gao M, Xie Y, Wang S, Shang S, Zhao J, Lu X (2021) A wideband picosecond pulsed electric fields (psPEF) exposure system for the nanoporation of biological cells. Bioelectrochemistry 140:107790. doi:10.1016/j.bioelechem.2021.107790 Schoenbach KH, Beebe SJ, Buescher ES (2001) Intracellular effect of ultrashort electrical pulses. Bioelectromagnetics 22 (6):440-448. doi:10.1002/bem.71 Ibrahimi N, Vallet L, Andre FM, Ariztia L, Rivaletto M, de Ferron AS, Novac BM, Mir LM, Pécastaing L (2020) A Subnanosecond Pulsed Electric Field System for Studying Cells Electropermeabilization. Ieee T Plasma Sci 48 (12):4242-4249. doi:10.1109/Tps.2020.3034286 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6486690","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":458933619,"identity":"a76def32-1725-4c57-81c5-ede041c122a0","order_by":0,"name":"Mackenzie Tardif-Kunk","email":"","orcid":"","institution":"Ellmer College of Health Sciences, Old Dominion University","correspondingAuthor":false,"prefix":"","firstName":"Mackenzie","middleName":"","lastName":"Tardif-Kunk","suffix":""},{"id":458933620,"identity":"bcff1eb6-bdf1-4993-a27d-48e541e05707","order_by":1,"name":"Martina Zamponi","email":"","orcid":"","institution":"Macon \u0026 Joan Brock Virginia Health Sciences Eastern Virginia Medical School At Old Dominion University","correspondingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Zamponi","suffix":""},{"id":458933621,"identity":"a683ec2f-21f2-4d20-b804-57d0b41bfcfd","order_by":2,"name":"Emily Old","email":"","orcid":"","institution":"Old Dominion University","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"","lastName":"Old","suffix":""},{"id":458933622,"identity":"d3b4a6c0-f4e4-4108-9c52-cafbf3484365","order_by":3,"name":"Augustine U. Anthony","email":"","orcid":"","institution":"Ellmer College of Health Sciences, Old Dominion University","correspondingAuthor":false,"prefix":"","firstName":"Augustine","middleName":"U.","lastName":"Anthony","suffix":""},{"id":458933623,"identity":"e1a98a7e-c32d-4d1a-8684-878285a42e2e","order_by":4,"name":"Chandler Knox","email":"","orcid":"","institution":"Ellmer College of Health Sciences, Old Dominion University","correspondingAuthor":false,"prefix":"","firstName":"Chandler","middleName":"","lastName":"Knox","suffix":""},{"id":458933624,"identity":"7c95e10e-050f-410f-810d-48bbdc43642b","order_by":5,"name":"Thomas R. Campbell","email":"","orcid":"","institution":"Ellmer College of Health Sciences, Old Dominion University","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"R.","lastName":"Campbell","suffix":""},{"id":458933625,"identity":"d9b07018-8281-4106-8260-387cd8408cd9","order_by":6,"name":"Ross A. Petrella","email":"","orcid":"","institution":"North Carolina State University","correspondingAuthor":false,"prefix":"","firstName":"Ross","middleName":"A.","lastName":"Petrella","suffix":""},{"id":458933626,"identity":"4a5aa6db-49c4-44a8-ad80-a1c8a0831963","order_by":7,"name":"Patrick C. Sachs","email":"","orcid":"","institution":"Ellmer College of Health Sciences, Old Dominion University","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"C.","lastName":"Sachs","suffix":""},{"id":458933627,"identity":"90623f23-ecc1-4039-a5a5-23dc350786df","order_by":8,"name":"Robert D. Bruno","email":"","orcid":"","institution":"Ellmer College of Health Sciences, Old Dominion University","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"D.","lastName":"Bruno","suffix":""},{"id":458933630,"identity":"997d4d16-c613-4880-8d1b-549a96442fe4","order_by":9,"name":"Peter A. Mollica","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYHACxgMPDBgY+BkYgCQDM3F6DiQAFUs2kKYFSBgcIFaLudjhAwcSCuzkjY83b5NgqLBObCCkxXJ2WgLQYcmG284cK5NgOJNOWIvB7RwDoJYDjNtu5JhJMLYdJl6L/eYZIC3/SNCSuEECpKWBCC0wvyTPOHOs2CLhWLoxQS3m0skHH3z4Y2fb39688caHGmtZwg5D4SUQUo6pZRSMglEwCkYBNgAAIwxCp0gzVv0AAAAASUVORK5CYII=","orcid":"","institution":"Ellmer College of Health Sciences, Old Dominion University","correspondingAuthor":true,"prefix":"","firstName":"Peter","middleName":"A.","lastName":"Mollica","suffix":""}],"badges":[],"createdAt":"2025-04-19 21:53:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6486690/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6486690/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83213187,"identity":"ac287014-9fcf-47d2-b593-9a92d3230c72","added_by":"auto","created_at":"2025-05-21 08:43:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":233719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of neural stem cells derived from an HD patient. (A)\u003c/strong\u003e Immunofluorescent labeling confirms neuroectodermal identity of HD-NSCs. Cells stained for nestin (green) and SOX2 (red) show robust expression with DAPI counterstain (blue), consistent with undifferentiated neural stem cell phenotype. PAX6 (green) and SOX1 (red) staining supports forebrain lineage specification. Scale bar = 100 µm. \u003cstrong\u003e(B)\u003c/strong\u003eTriplet-primed PCR followed by capillary electrophoresis confirms CAG repeat expansions of 18 and 44 in the HTT gene (blue peaks), consistent with a heterozygous HD genotype. Red peaks represent MapMarker® 1000 (ROX), used for internal fragment calibration.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6486690/v1/bd7179d04f13b0c6b57f4148.png"},{"id":83213190,"identity":"938dc5f4-755f-4e4d-82ad-293a6f29080b","added_by":"auto","created_at":"2025-05-21 08:43:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":211878,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epsPEF exposure does not compromise HD-NSC viability. (A)\u003c/strong\u003eRepresentative images of HD- NSCs stained for live cells (calcein-AM, green) and dead cells (BOBO-3 iodide, red) 30 minutes after psPEF exposure at 20 or 40 kV/cm. Scale bar = 200 µm. \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of live/dead cell ratios. Data represent mean ± SEM from three independent experiments (three images per replicate). Statistical comparisons were made using one-way ANOVA with Tukey’s post hoc test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6486690/v1/ad17b60c4f0a8e1ffd7b0b7e.png"},{"id":83213192,"identity":"b0967c90-e3d3-47d3-a71c-75386faf9ce5","added_by":"auto","created_at":"2025-05-21 08:43:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":227892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epsPEF reduces huntingtin aggregation in HD-NSCs. (A)\u003c/strong\u003e Representative confocal images of HD-NSCs stained for PolyQ (green), HTT (red), and DAPI (blue) at 30 minutes and 24 hours post-psPEF exposure (20 and 40 kV/cm). Colored arrows indicate regions of aggregate accumulation. Scale bar = 10 µm. \u003cstrong\u003e(B-E)\u003c/strong\u003e Quantification of: \u003cstrong\u003e(B)\u003c/strong\u003eaggregate count per cell, \u003cstrong\u003e(C)\u003c/strong\u003e aggregate area per cell, \u003cstrong\u003e(D)\u003c/strong\u003eaverage aggregate size, and \u003cstrong\u003e(E)\u003c/strong\u003e aggresomes per cell (defined as aggregates \u0026gt;4 µm²). Data represent mean ± SEM from ≥9 images per condition, analyzed with AggreCount. Statistical comparisons were made using one-way ANOVA with Tukey’s post hoc test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6486690/v1/f57cbb572276d442fe43632c.png"},{"id":83214343,"identity":"a5a6bbb3-7c52-47ed-b9e9-e449cdb1e4af","added_by":"auto","created_at":"2025-05-21 08:51:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53247,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epsPEF modulates subcellular localization and size of PolyQ aggregates in HD-NSCs.\u003c/strong\u003e\u003cbr\u003e\nBar graphs show quantification of PolyQ-positive aggregates in cytosolic \u003cstrong\u003e(A\u003c/strong\u003e,\u003cstrong\u003eB)\u003c/strong\u003e, perinuclear \u003cstrong\u003e(C\u003c/strong\u003e, \u003cstrong\u003eD)\u003c/strong\u003e, and nuclear \u003cstrong\u003e(E\u003c/strong\u003e, \u003cstrong\u003eF)\u003c/strong\u003ecompartments following exposure to 0, 20, or 40 kV/cm psPEF. Panels A, C, and E represent the average number of aggregates per cell; Panels B, D, and F display corresponding aggregate area per cell in each compartment. \u003cstrong\u003e(G)\u003c/strong\u003eDistribution of total aggregates across subcellular compartments (cytosol, perinuclear, and nucleus) for each treatment condition. Data represent mean ± SEM from at least 9 non-overlapping images per group, derived from three independent experiments. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test. Asterisks indicate significance relative to the no-pulse control: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6486690/v1/8d10d93ec3ba872d71bf534e.png"},{"id":83215280,"identity":"f42fcb4a-8cc6-4c35-a5f7-c6cd9605c9ed","added_by":"auto","created_at":"2025-05-21 08:59:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":27601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epsPEF decrease expression of CACNA1C and PAX independent of HTT. \u003c/strong\u003eQuantification of HTT, CACNA1C, and PAX6 mRNA expression in HD-NSCs exposed to psPEF relative to the non-pulsed control (NPC). Data is presented as the mean fold change per RT-qPCR reaction ± SEM. Comparison was performed by one-way ANOVA with Tukey’s post hoc (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6486690/v1/d37abb4b08791252ad083d0f.png"},{"id":83213194,"identity":"999e2bb3-2e62-410e-b515-1006265d1e96","added_by":"auto","created_at":"2025-05-21 08:43:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":214934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epsPEF-induced modulation of mitochondrial membrane potential in HD neural stem cells.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eHD-NSCs were exposed to picosecond pulsed electric fields (psPEF) at 0, 20, or 40 kV/cm. Mitochondrial membrane potential was assessed using TMRE (50 nM), with live-cell confocal imaging performed every 5 minutes for 30 minutes post-treatment. A fire LUT was applied post-analysis (ImageJ) to visualize intensity differences, with yellow/green indicating high membrane potential and blue indicating depolarization. LUT was applied only after quantitative analysis. Scale bar = 75 μm. \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of TMRE fluorescence intensity over time. Data represent integrated signal from ≥6 ROIs per condition (10–20 cells per ROI), processed using a standardized ImageJ macro with Otsu thresholding. Values shown as min and max values. Although not statistically significant—no statistically significant differences were detected by two-way repeated measures ANOVA with Tukey’s post hoc test, cells treated with 40 kV/cm exhibited a trend toward increased mitochondrial polarization, suggesting a possible non-lethal bioenergetic response to psPEF.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6486690/v1/eaf71f14f61b682aac933624.png"},{"id":87500374,"identity":"cc726f92-9ad5-481c-ae4c-88930faf701a","added_by":"auto","created_at":"2025-07-24 13:46:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1934442,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6486690/v1/9f19f6dc-b82c-4bb0-a2fe-c8135de89775.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Picosecond Pulsed Electric Field-Induced Disaggregation of Polyglutamine Aggregates in Huntington’s Disease Neural Stem Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuntington\u0026rsquo;s Disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats within exon 1 of the Huntingtin (\u003cem\u003eHTT\u003c/em\u003e) gene [1,2]. When this repeat exceeds approximately 35 units, the translated huntingtin protein (HTT) contains a pathogenic polyglutamine (PolyQ) tract that disrupts its native conformation, promoting the formation of β-sheet-rich amyloid fibrils. These protein aggregates accumulate in both the cytoplasm and nucleus, interfering with essential cellular processes such as transcription, proteostasis, and axonal transport [3\u0026ndash;5]. In HD, aggregate toxicity disproportionately affects medium spiny neurons (MSNs) of the striatum, which are critical for motor coordination and cognitive function [6,4]. The progressive degeneration of MSNs leads to the hallmark triad of HD symptoms: involuntary movements, cognitive decline, and psychiatric disturbances. Despite improved understanding of HD pathophysiology, there remains no approved treatment that modifies disease progression or reduces aggregate burden [7].\u003c/p\u003e \u003cp\u003eProtein aggregation is increasingly recognized as a convergent pathological feature across neurodegenerative diseases, including Alzheimer\u0026rsquo;s, Parkinson\u0026rsquo;s, and Huntington\u0026rsquo;s disease [8\u0026ndash;14]. Therapeutic efforts to target aggregated proteins have traditionally relied on small molecules, antibodies, or gene-silencing approaches [15\u0026ndash;26]. However, these methods often suffer from limited bioavailability, poor blood\u0026ndash;brain barrier penetration, off-target effects, or suboptimal clearance of established aggregates. Alternative physical modalities that non-invasively modulate protein conformation or cellular pathways represent a promising but underexplored strategy.\u003c/p\u003e \u003cp\u003ePulsed electric fields (PEFs) have been widely studied for their ability to modulate cellular behavior through controlled electrical stimulation [27,28]. Conventional applications of PEFs in the microsecond to nanosecond range include reversible electroporation for gene delivery, irreversible electroporation for tissue ablation, and nanosecond PEFs (nsPEFs) for subcellular manipulation and cell death induction [29\u0026ndash;35]. The biological effects of these pulses are heavily dependent on pulse duration, intensity, rise time, and the number of delivered pulses [36\u0026ndash;43]. Among these, picosecond pulsed electric fields (psPEF) are the least characterized yet potentially the most selective modality for intracellular targeting. PsPEFs are defined by ultra-short pulse durations (\u0026lt;\u0026thinsp;1 ns) with subnanosecond rise times, which allow them to traverse the cell membrane without inducing membrane permeabilization due to their operation below the plasma membrane charging time constant (~\u0026thinsp;75 ns) [44\u0026ndash;46,31,30]. This property enables direct coupling of psPEFs to cytosolic components while minimizing collateral membrane damage.\u003c/p\u003e \u003cp\u003eEmerging theoretical and experimental evidence suggests that psPEFs can act as a biophysical modulator of protein structure [47,48,31]. Molecular dynamics simulations have demonstrated that intense picosecond electric fields can induce a conformational shift in amyloidogenic proteins, such as β-amyloid and α-synuclein, favoring transitions from β-sheet to α-helical structures [49,50]. This mechanism has been proposed to underlie electric field-mediated disaggregation of protein fibrils. Experimental studies using purified protein models support this hypothesis, showing that localized electric fields can fragment amyloid plaques or prevent fibrillization [51,52]. However, no study to date has investigated psPEF effects on protein aggregates within a human disease-relevant neural cell context. This constitutes a critical gap in the translation of psPEF technology from a physical or biochemical phenomenon to a potential therapeutic tool.\u003c/p\u003e \u003cp\u003eTo address this, we applied psPEF to human neural stem cells derived from an HD patient to evaluate whether this non-invasive modality can modulate disease-relevant phenotypes, including polyglutamine aggregation and mitochondrial dysfunction. We utilized neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) reprogrammed from HD patient fibroblasts. These cells endogenously express full-length mutant huntingtin under native regulatory control and recapitulate early molecular features of HD, including PolyQ aggregation, altered calcium homeostasis, and mitochondrial dysfunction [53\u0026ndash;58]. While mature neurons represent the ultimate target of neurodegenerative pathology, NSCs provide a scalable, tractable, and physiologically relevant model system for initial mechanistic studies. Their rapid expansion, consistent phenotype, and preservation of disease-relevant stress pathways make them ideally suited for high-content analysis of psPEF-induced biophysical and molecular responses. Moreover, early-stage investigation in NSCs enables controlled, within-line comparisons to quantify psPEF-mediated changes in aggregate burden, gene expression, and mitochondrial membrane potential (ΔΨm) without confounding variation from heterogeneous differentiation states. This approach establishes a foundational framework for future studies in differentiated neurons and more complex multicellular models.\u003c/p\u003e \u003cp\u003eTo ensure experimental tractability while capturing both immediate and intermediate responses to psPEF, we focused our analysis on 30-minute and 24-hour post-treatment time points. This timeframe was strategically chosen to detect acute changes in aggregate morphology, transcriptional modulation, and mitochondrial dynamics while avoiding variability introduced by extended in vitro culture. Although longer-term effects were not assessed in this study, these intervals provide critical insight into the initial cellular response to psPEF stimulation and form the basis for future investigations into the persistence and downstream impact of psPEF-mediated modulation. Field strengths of 20 and 40 kV/cm were selected based on previous bioelectric studies demonstrating intracellular modulation without overt membrane damage at these intensities [30,59,38,60]. These values represent physiologically relevant upper and mid-range intensities capable of modulating intracellular targets without inducing cellular toxicity.\u003c/p\u003e \u003cp\u003eTo deliver psPEF with spatial and temporal precision, we employed a custom-engineered 3D bioprinter platform equipped with a coaxial electrode probe connected to a picosecond pulse generator [61,59]. Using computational modeling and precise motion control, we exposed HD-NSCs to 1.8k pulses at electric field strengths of 20 kV/cm and 40 kV/cm. We then assessed mHTT aggregation, gene expression, and mitochondrial function at multiple time points post-exposure. This work represents the first \u003cem\u003ein vitro\u003c/em\u003e demonstration of psPEF-mediated disaggregation of pathological protein aggregates in a human neural stem cell model of HD and provides foundational evidence for the use of psPEF as a non-invasive bioelectronic approach to proteopathy modulation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Lines and Culture\u003c/h2\u003e \u003cp\u003eThe neuronal stem cell (NSC) lines used in this study were established in our laboratory from human HD patient-derived fibroblasts (GM04022; Coriell Institute) that had previously been reprogrammed to induced pluripotent stem cells (iPSCs) and have been phenotypically validated in earlier work [62,63]. HD-NSCs were cultured in STEMdiff\u0026trade; Neural Progenitor Medium (Stemcell Technologies) supplemented with 1% antibiotic-antimycotic solution (Life Technologies) and maintained at 37\u0026deg;C in a humidified 5.0% CO₂ atmosphere. Media were replaced every 48 to 72 hours to ensure optimal nutrient availability and minimize metabolic stress. Cells were seeded onto Geltrex\u0026trade;-coated 6-well plates (1:100 dilution, Thermo Fisher Scientific) and passaged using TrypLE Express (Gibco) following the manufacturer\u0026rsquo;s protocol. All experiments were performed using early-passage cultures to minimize phenotype drift.\u003c/p\u003e \u003cp\u003eFor psPEF exposure and imaging-based analyses, a center-seeding method was used to standardize the exposure area. Geltrex\u0026trade; (50\u0026ndash;75 \u0026micro;L per well) was applied only to the center of each culture surface and incubated for 1 hour. Cells were then seeded directly onto this coated region at densities described per assay, followed by the addition of full medium volume after 1 hour. This technique is referred to herein as \u0026ldquo;center-seeding.\u0026rdquo;\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCharacterization of iPSC-Derived Neural Stem Cells\u003c/h3\u003e\n\u003cp\u003eTo confirm NSC identity and maintenance of neural progenitor characteristics, immunocytochemistry (ICC) was performed on adherent cultures. Cells were seeded at 3.5\u0026times;10⁴ per well in Geltrex\u0026trade;-coated 8-well chamber slides and fixed after 48 hours using 10% neutral-buffered formalin. Permeabilization was carried out using 0.1% NP-40 in PBS for 10 minutes, followed by blocking with 10% normal goat serum for 1 hour.\u003c/p\u003e \u003cp\u003ePrimary antibodies targeting canonical NSC markers were applied in PBS containing 1% goat serum for 1 hour at room temperature. These included SOX1 (ab87775, 1:200, Abcam), SOX2 (ab97959, 1:1000, Abcam), and PAX6 (13B10-1A10, 1:200, Thermo Fisher Scientific) to confirm neuroectodermal lineage commitment, and nestin (MA1-110, 1:100, Thermo Fisher Scientific) to assess intermediate filament expression. After three PBS washes, cells were incubated with Alexa Fluor-conjugated secondary antibodies (488 and 568; 1:1000, Thermo Fisher) for 1 hour. DAPI (1:1000) was used to counterstain nuclei. Slides were mounted with Fluoromount-G\u0026reg; and imaged using a Zeiss Axio Observer Z1 inverted microscope under standardized acquisition settings.\u003c/p\u003e\n\u003ch3\u003eMutant HTT Fragment Analysis\u003c/h3\u003e\n\u003cp\u003eTo confirm the presence of expanded CAG repeats in \u003cem\u003eHTT\u003c/em\u003e, triplet-primed PCR (TP-PCR) followed by capillary electrophoresis was performed using previously validated protocols [64,62]. Fluorescently labeled primers targeting the HTT locus were used, and PCR amplification was carried out with Platinum\u0026trade; Taq High Fidelity polymerase under optimized conditions. Amplicons were denatured and separated using a 3130 Genetic Analyzer (Applied Biosystems). Size distributions were analyzed with GeneMarker\u0026reg; v2.6.7 using custom bin definitions created from Coriell reference HD cell lines.\u003c/p\u003e\n\u003ch3\u003epsPEF Delivery Using 3D Printer-Based Electrode System\u003c/h3\u003e\n\u003cp\u003ePicosecond pulsed electric fields (psPEF) were delivered using a custom-designed in vitro stimulation platform that integrates a high-speed pulse generator with a programmable 3D motion control system. The exposure system was based on a modified FELIX 3.0 3D printer (FELIXrobotics, Ijsselstein, Netherlands) equipped with a coaxial electrode probe mounted to the print head, enabling spatially controlled non-contact stimulation of cell monolayers [59].\u003c/p\u003e \u003cp\u003eThe electrode probe consisted of parallel tungsten wires (0.1 mm diameter), each measuring approximately 1.5 cm in length. The wires were soldered to the signal and ground terminals of a 50 Ω coaxial transmission line and embedded in epoxy insulation, leaving 1.0 cm of exposed tungsten at the tip. Electrodes were positioned 1 mm apart (edge-to-edge), forming a narrow, high-field fringing configuration. The probe was mounted to the X-axis carriage and vertically calibrated using the mechanical pitch of the printer\u0026rsquo;s leadscrew to maintain a consistent 200 \u0026micro;m (0.2 mm) standoff from the culture surface.\u003c/p\u003e \u003cp\u003eHigh-voltage pulse generation was provided by an FPG 10-10PM1 pulse generator (FID GmbH, Burbach, Germany), capable of producing unipolar Gaussian pulses with amplitudes up to 10 kV into a 50 Ω load. For this study, the pulse amplitude was adjusted to 2.0 kV and 4.0 kV, corresponding to calculated electric field strengths of 20 kV/cm and 40 kV/cm at the cell surface. Each pulse exhibited a rise time of 250\u0026ndash;350 ps and a full-width at half-maximum (FWHM) duration of 660 ps, depending on the voltage setting. Pulses were delivered at a fixed repetition rate of 1 kHz, consistent with the generator\u0026rsquo;s internal triggering configuration. The pulse repetition frequency of 1 kHz was chosen based on common practice within ultrashort pulse bioelectric studies to balance effective stimulation with minimal thermal accumulation [39,65]. Prior studies employing similar ultrashort pulses at frequencies around 1 kHz have demonstrated effective intracellular modulation without observable thermal or cytotoxic effects. Field strength calibration was verified through direct voltage measurements and confirmed using FDTD simulations in CST Microwave Studio.\u003c/p\u003e \u003cp\u003eCells were exposed to a total of approximately 1,800 pulses as the probe traversed an S-shaped scan path generated using custom MATLAB scripts converted to G-code. The spatial pulse delivery ensured uniform treatment across the designated cell zone without overlapping tracks. Prior to pulse delivery, culture medium was removed and replaced with PBS to reduce dielectric damping and avoid field dispersion caused by high ionic conductivity.\u003c/p\u003e \u003cp\u003eThis 3D bioprinter-based psPEF delivery system allows for highly reproducible, spatially uniform stimulation under tightly controlled electrical and mechanical parameters, enabling novel investigations of subcellular responses to non-contact ultrashort electric fields. All analyses were performed at 30 minutes and 24 hours post-exposure, selected to capture both acute and intermediate cellular responses to psPEF while minimizing variability associated with prolonged \u003cem\u003ein vitro\u003c/em\u003e culture.\u003c/p\u003e\n\u003ch3\u003eCell Viability Assessment\u003c/h3\u003e\n\u003cp\u003eCell viability following psPEF exposure was assessed using the LIVE/DEAD\u0026trade; Cell Imaging Kit (Thermo Fisher Scientific), which distinguishes viable cells via intracellular esterase activity (calcein AM, green fluorescence) and dead cells by membrane-impermeable BOBO-3\u0026trade; iodide (red fluorescence). HD-NSCs were center-seeded at 3\u0026times;10⁴ cells per well in Geltrex\u0026trade;-coated 12-well plates and incubated for 48 hours before psPEF exposure at 0, 20, or 40 kV/cm. After pulsing, cells were allowed to recover in their respective culture media for 30 minutes.\u003c/p\u003e \u003cp\u003eFollowing recovery, the LIVE/DEAD\u0026trade; reagents were added directly to the culture medium at a 1:1 dilution and incubated at room temperature for 15 minutes in the dark. Cells were then washed with PBS to remove excess dye and returned to dye-free media. Fluorescent images were captured using a Zeiss Axio Observer Z1 inverted microscope with consistent settings across all conditions. A total of twelve images per treatment group (three per well from four replicates) were collected from non-overlapping fields of view.\u003c/p\u003e \u003cp\u003eViable and non-viable cells were manually counted by a blinded observer using ImageJ. The percentage of live cells per field was calculated and averaged across replicates. Group differences were analyzed as described in the Statistical Analysis section. This method allowed for the assessment of cytotoxic effects of psPEF under the tested conditions while minimizing observer bias and ensuring standardized acquisition.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry and Confocal Imaging\u003c/h2\u003e \u003cp\u003eFor aggregate quantification, human HD neural stem cells (NSCs) were seeded at 2 \u0026times; 10⁴ cells per dish onto Geltrex\u0026trade;-coated glass-bottom dishes (MatTek; 35-mm dish, No. 1.5 coverslip, 10 mm glass diameter) using the center-seeding method. Cells were allowed to adhere for 48 hours prior to psPEF exposure. Following stimulation at 0, 20, or 40 kV/cm, cells were fixed at 30 minutes or 24 hours post-treatment using ice-cold 100% methanol at \u0026minus;\u0026thinsp;20\u0026deg;C for 10 minutes. Fixed cells were blocked with 10% normal goat serum in PBS for 1 hour and stained with primary antibodies against polyglutamine-expanded proteins (PolyQ; Sigma MAB1574, 1:1000) and huntingtin (HTT; Abcam ab109115, 1:1000), diluted in 1% goat serum in PBS for 1 hour at room temperature. Secondary antibodies conjugated to Alexa Fluor 488 and 568 (Thermo Fisher, 1:1000) were used for detection. DAPI was used for nuclear counterstaining. Samples were mounted in Fluoromount-G\u0026reg; and sealed for imaging.\u003c/p\u003e \u003cp\u003eConfocal imaging was performed on a Leica Stellaris 5 system equipped with a DMI8 inverted microscope, HyD detectors, and a tunable white light laser. Imaging was conducted using a 40x oil immersion objective (NA 1.40). Acquisition settings were rigorously standardized across all experimental conditions, including a 1024 \u0026times; 1024-pixel resolution, a z-step size of 0.3 \u0026micro;m, pinhole set to 1 Airy unit, and laser power, detector gain, and offset fixed across all sessions after optimization on control samples. To reduce noise and enhance signal fidelity, line averaging (3\u0026ndash;4\u0026times;) and frame accumulation (2\u0026times;) were applied. At least ten non-overlapping fields of view were imaged per condition, each containing 10\u0026ndash;20 cells. All imaging was performed in a blinded and randomized fashion.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAggregate Quantification\u003c/h3\u003e\n\u003cp\u003eQuantitative image analysis of PolyQ aggregates was performed using Fiji (ImageJ v1.53) and the AggreCount macro was specifically chosen due to its automated, objective, and reproducible quantification of aggregates [66]. This approach eliminated subjective interpretation and minimized potential observer bias, ensuring robust, reproducible data irrespective of operator experience. Prior to analysis, raw confocal z-stacks were converted to 16-bit grayscale and projected into two-dimensional images using the \u0026ldquo;sum slices\u0026rdquo; function to preserve intensity values across all z-planes. This approach ensured uniform image input and was well-suited to detecting small, high-intensity puncta consistent with protein aggregates.\u003c/p\u003e \u003cp\u003eThe AggreCount macro was run in segmentation-based \u0026ldquo;cell processing\u0026rdquo; mode using validated parameters. Thresholding was manually calibrated using non-pulsed control samples to ensure consistent background exclusion across all groups. Aggregates were segmented using a minimum area threshold of 0.2 \u0026micro;m\u0026sup2; and a maximum of 20 \u0026micro;m\u0026sup2;. Aggregates exceeding 4.0 \u0026micro;m\u0026sup2; were classified as \u0026ldquo;aggresomes.\u0026rdquo; Perinuclear localization was defined as aggregate proximity within 10 pixels of the nuclear boundary. The plugin was configured to exclude nuclei smaller than 50 \u0026micro;m\u0026sup2; and cells smaller than 75 \u0026micro;m\u0026sup2;; both cell and nuclear segmentation used a strictness value of 5 to ensure edge fidelity and minimize overlap artifacts.\u003c/p\u003e \u003cp\u003eQuantitative outputs included aggregate count, mean aggregate size, total aggregate area, aggresome count, and compartment-specific distribution (cytosolic, perinuclear, nuclear) normalized per nucleus. All analyses were conducted using batch processing to maintain parameter consistency. Fluorescence localization was verified using orthogonal slice views and maximum intensity projections to confirm the spatial accuracy of aggregate classification. Comparisons between treatment groups and localization categories were conducted as outlined in the Statistical Analysis section.\u003c/p\u003e\n\u003ch3\u003eQuantitative RT-PCR\u003c/h3\u003e\n\u003cp\u003eHD-NSCs were center-seeded on Geltrex\u0026trade; in 12-well plates at a density of 4x104 cells. Cells were pulsed with 4 replicates at 20 kV/cm or 40 kV/cm or designated as a non-pulsed control. Total RNA was isolated 24 hours after pulsing using TRIzol (Invitrogen) according to the manufacturer\u0026rsquo;s protocol. RNA quantity and quality were determined by the absorbance at 260/280 nm using a NanoDrop 2000 (Thermo Fisher). Any genomic DNA was removed from the RNA sample by deoxyribonuclease I (Invitrogen) and the manufacturer\u0026rsquo;s protocol. The High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used to synthesize cDNA from the RNA samples (1 mg). Quantitative RT-PCR was performed on a QuantStudioTM 7 Flex Real-Time PCR System (Applied Biosystems). TaqMan Gene Expression Assays (Applied Biosystems) were used to quantify mRNA for the following genes: HTT (Hs00918174_m1), CACNA1C (Hs00167681_m1), and PAX6 (Hs00240871_m1). ACTB (Hs01060665_g1) served as the endogenous housekeeping gene. All samples were analyzed in triplicate using TaqMan Fast Advanced Master Mix (Life Technologies) and 5 ng of cDNA per reaction according to the manufacturer\u0026rsquo;s protocol. The 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method was used to calculate the fold-changes relative to the no-pulse control (NPC) [67]. Group differences in gene expression were evaluated as described in the Statistical Analysis section.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTMRE Imaging and Analysis\u003c/h2\u003e \u003cp\u003eHD-NSCs were center-seeded onto Geltrex\u0026trade;-coated 35 mm glass-bottom dishes (MatTek; No. 1.5 coverslip, 10-mm glass area) at a density of 2\u0026times;10⁴ cells per dish and incubated for 48 hours prior to psPEF exposure. After pulsing at 0, 20, or 40 kV/cm, cells were immediately incubated in pre-warmed media containing 50 nM tetramethylrhodamine ethyl ester (TMRE; Thermo Fisher Scientific), a cell-permeant, cationic dye that accumulates in polarized mitochondria. Following 30 minutes of incubation under standard conditions (37\u0026deg;C, 5% CO₂), cells were washed twice with PBS and returned to fresh medium without TMRE prior to imaging.\u003c/p\u003e \u003cp\u003eLive imaging was performed every 5 minutes for 30 minutes using a Leica Stellaris 5 confocal microscope with a DMI8 inverted platform, HyD detectors, and a 40x oil immersion objective (NA 1.40). TMRE was excited at 549 nm, and emission was collected at 574 nm. Acquisition parameters\u0026mdash;including laser power, gain, detector settings, and pinhole diameter\u0026mdash;were held constant across all samples and time points. Z-stacks were collected with identical slice numbers and spacing, allowing consistent volumetric fluorescence comparison across conditions.\u003c/p\u003e \u003cp\u003eImage analysis was conducted using a custom macro in Fiji (ImageJ v1.53). Each z-stack image was converted to 8-bit grayscale and thresholded using Otsu\u0026rsquo;s method to isolate TMRE-positive signal. [68] A binary mask was applied, and integrated fluorescence intensity was quantified within the thresholded region using the \"Analyze Particles\" function. No manual ROI selection, background subtraction, or filtering was applied to preserve consistency.\u003c/p\u003e \u003cp\u003eEach condition was represented by at least six replicate regions of interest (ROIs), each containing approximately 10\u0026ndash;20 cells. The same fields of view were tracked over the 30-minute time course. Raw values were used for statistical comparison. Time- and treatment-related effects were analyzed using the approach described in the Statistical Analysis section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism v10.4.1. For all parametric comparisons involving three or more groups, one-way analysis of variance (ANOVA) was used followed by Tukey\u0026rsquo;s post hoc test to evaluate pairwise differences. This approach was applied to quantitative RT-PCR data, viability assays, and aggregate burden metrics across treatment conditions. For comparisons involving categorical variables such as subcellular aggregate localization, chi-squared analysis was employed to assess distributional shifts across compartments.\u003c/p\u003e \u003cp\u003eTime-course data from TMRE imaging were analyzed using a two-way repeated measures ANOVA to evaluate the effects of time and treatment on mitochondrial membrane potential. Assumptions of normality and sphericity were assessed using the Shapiro-Wilk and Mauchly\u0026rsquo;s tests, respectively. In instances where assumptions were violated, nonparametric alternatives including the Friedman test and aligned rank transform were applied. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Where statistical thresholds were not met, biologically relevant trends were reported when supported by effect size, directionality, and consistency across replicates. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation unless otherwise noted.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of Neural Stem Cells by Immunocytochemistry and Genotyping\u003c/h2\u003e \u003cp\u003eTo verify the identity and regional specification of the iPSC-derived HD neural stem cells (NSCs), immunocytochemistry was performed to assess expression of key neurodevelopmental markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). SOX1 and SOX2, transcription factors associated with neuroectodermal lineage commitment and maintenance of NSC identity, exhibited strong nuclear localization in the majority of cells. PAX6, a regulator of forebrain patterning, was similarly localized to the nucleus in a broad subset of cells, consistent with successful differentiation toward a dorsal forebrain lineage. Nestin, a marker of intermediate filaments in proliferating NSCs, displayed filamentous cytoplasmic staining throughout the culture. These patterns confirm a neuroepithelial-like NSC phenotype, supportive of subsequent differentiation into central nervous system lineages.\u003c/p\u003e \u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e here]\u003c/p\u003e \u003cp\u003eTo confirm our previous characterization of the HD genotype of the NSC line (GM04022), triplet-primed PCR (TP-PCR) followed by capillary electrophoresis was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) [63]. Fragment analysis revealed heterozygous CAG repeat lengths of 18 and 44, consistent with a pathogenic HD allele and a normal allele. These results verify that the cell line retains the genetic hallmark of Huntington\u0026rsquo;s disease and is suitable for modeling polyglutamine pathology in vitro.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003epsPEF Exposure Does Not Compromise Cell Viability\u003c/h2\u003e \u003cp\u003eGiven that PEFs can induce membrane permeabilization or cell death depending on pulse parameters, we evaluated the cytotoxic potential of psPEF exposure using a LIVE/DEAD\u0026trade; viability assay. HD-NSCs were exposed to 20 or 40 kV/cm psPEF and assessed 30 minutes post-exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Live cells were defined by intracellular esterase activity (calcein AM), while non-viable cells were labeled by membrane-impermeant BOBO-3\u0026trade; iodide. No statistically significant differences in viability were detected at either field strength when compared to non-pulsed controls (20 kV/cm: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.536; 40 kV/cm: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.475), indicating that psPEF treatment under these conditions does not compromise membrane integrity or induce acute cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB)​.\u003c/p\u003e \u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e here]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003epsPEF Reduces mHTT Aggregate Burden in HD-NSCs\u003c/h2\u003e \u003cp\u003eTo determine whether psPEF could influence protein aggregation, HD-NSCs were immunoassayed for mutant huntingtin (PolyQ) following exposure to 0, 20, or 40 kV/cm psPEF. Compared to untreated controls, psPEF-treated cells exhibited visibly reduced PolyQ puncta intensity and size at both 30 minutes and 24 hours post-exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Confocal image stacks were analyzed using the AggreCount macro to quantify total PolyQ-positive puncta, average aggregate size, and aggresome burden.\u003c/p\u003e \u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e here]\u003c/p\u003e \u003cp\u003eInterestingly, despite significant reductions in aggregate size and aggresome load, the total number of aggregates per cell did not significantly decrease at either field strength (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This observation may suggest a remodeling or fragmentation of larger aggregates into smaller subunits rather than complete clearance. Thus, the psPEF-induced disaggregation may primarily reduce aggregate pathogenicity by decreasing their size and altering intracellular distribution, rather than eliminating aggregate formation entirely.\u003c/p\u003e \u003cp\u003eHowever, the average aggregate area per cell decreased by 66% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0174) and 63% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0224) for 20 and 40 kV/cm, respectively, 30 minutes after pulsing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These reductions were further amplified at 24 hours, with aggregate areas reduced by 74% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0049) and 77% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0052) for 20 and 40 kV/cm, respectively. Similarly, average individual aggregate size decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) by 58\u0026ndash;69% across both field strengths and time points (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0193, 0.0092, 0.0196, 0.0058, respectively). The number of aggresomes per cell (\u0026ge;\u0026thinsp;4.0 \u0026micro;m\u0026sup2;) was also significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE)\u0026mdash;by up to 56% at 30 minutes and 43% at 24 hours (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0076, respectively). Aggregate segmentation parameters were calibrated using non-pulsed controls and visually validated against raw image stacks to confirm detection fidelity across subcellular compartments. This ensured consistent identification of small puncta and excluded background artifacts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSubcellular Distribution of PolyQ Aggregates Is Differentially Modulated by psPEF\u003c/h2\u003e \u003cp\u003eFollowing psPEF exposure, compartment-specific changes in aggregate burden were observed across cytosolic, perinuclear, and nuclear regions. In the cytosolic compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), both aggregate number and total area were significantly reduced at 30 minutes post-treatment. Aggregate counts declined by 59\u0026ndash;62% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and total aggregate area decreased by 79\u0026ndash;81% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0012, 0.0007, respectively) at both 20 and 40 kV/cm. These reductions persisted through 24 hours post-exposure, indicating a durable effect of psPEF on cytosolic aggregate load.\u003c/p\u003e \u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e here]\u003c/p\u003e \u003cp\u003eIn the perinuclear compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;D), psPEF exposure did not significantly alter aggregate number or area at 30 minutes post-treatment. However, a delayed 33% reduction in aggregate count was observed at 24 hours following 20 kV/cm stimulation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating a time-dependent effect on perinuclear aggregate clearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Notably, the total area of perinuclear aggregates per cell decreased by 62\u0026ndash;67% at 30 minutes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0139, 0.0217, respectively) and by 73\u0026ndash;74% at 24 hours (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0036, 0.0072, respectively), consistent with a sustained morphological remodeling of perinuclear aggregate burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eIn the nuclear compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), psPEF exposure led to a transient increase in aggregate number at 30 minutes (20 kV/cm: 42.6%, 40 kV/cm: 44.0%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), without a significant change in total aggregate area. This transient nuclear accumulation coincided with reductions in cytosolic and perinuclear aggregates.\u003c/p\u003e \u003cp\u003eThe overall distribution of aggregates across compartments is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG. In untreated cells, aggregates were predominantly cytosolic (45.2%), followed by perinuclear (40.0%) and nuclear (14.9%) localization. psPEF exposure shifted this balance toward the nucleus at 30 minutes, particularly at higher field strengths. This redistribution was partially reversed by 24 hours, suggesting a dynamic reorganization of aggregate localization rather than degradation alone. Pulse intensity did not significantly alter the direction of redistribution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGene Expression Changes Following psPEF Treatment\u003c/h2\u003e \u003cp\u003eQuantitative RT-PCR was performed 24 hours after psPEF exposure to evaluate whether observed changes in aggregate burden could be attributed to transcriptional regulation. No significant differences in HTT mRNA expression were observed (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.479 for 20 kV/cm; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.622 for 40 kV/cm). However, both PAX6 and CACNA1C transcripts were significantly downregulated following exposure, with more pronounced suppression at higher field strength. PAX6 levels declined by ~\u0026thinsp;26% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0195) at 40 kV/cm, while CACNA1C expression decreased by ~\u0026thinsp;23% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0493), suggesting that psPEF modulates the expression of neurodevelopmental and ion channel genes independently of HTT transcription​ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e here]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003epsPEF May Influence Mitochondrial Polarization\u003c/h2\u003e \u003cp\u003eGiven that mitochondrial dysfunction and reduced membrane potential are hallmark features of Huntington\u0026rsquo;s disease pathology, we assessed whether psPEF stimulation influences mitochondrial polarization in HD-NSCs. [69\u0026ndash;73] Cells were loaded with TMRE, a potentiometric dye that accumulates in mitochondria proportional to membrane potential (ΔΨm), and live-cell confocal imaging was performed over a 30-minute time course following psPEF exposure.\u003c/p\u003e \u003cp\u003e[Insert Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e here]\u003c/p\u003e \u003cp\u003eQualitative assessment of TMRE fluorescence revealed modest increases in signal intensity in psPEF-treated cells, particularly in the 40 kV/cm group, relative to non-pulsed controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Fluorescence enhancement appeared most pronounced at 5- and 30-minutes post-treatment, suggesting a delayed but potentially sustained bioenergetic response. These differences were visualized using a fire lookup table (LUT) applied post-quantification to highlight regional variations in signal.\u003c/p\u003e \u003cp\u003eQuantitative analysis of integrated TMRE intensity showed a trend toward elevated membrane potential in psPEF-treated groups compared to control, with the most consistent increase observed in the 40 kV/cm condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Although differences did not reach statistical significance in two-way repeated measures ANOVA, the directionality of the response\u0026mdash;especially given the known baseline mitochondrial depolarization in HD cells\u0026mdash;suggests that psPEF may influence mitochondrial function in a non-lethal and potentially restorative manner. These findings support further exploration of psPEF as a modulator of mitochondrial health in models of proteopathic stress.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe application of psPEF in biomedical research represents a novel frontier within bioelectrics, uniquely suited to modulate intracellular processes without irreversibly disrupting membrane integrity [31,30,60]. Unlike microsecond and nanosecond pulses\u0026mdash;commonly employed for electroporation, apoptosis induction, or electrochemotherapy\u0026mdash;psPEFs operate at subnanosecond timescales, allowing interaction with intracellular targets prior to plasma membrane charging. This study provides the first evidence that psPEF can non-lethally reduce mHTT aggregation and modulate mitochondrial polarization in a human cellular model of Huntington\u0026rsquo;s disease, supporting its potential as a bioelectronic approach to modulating HD-relevant intracellular stress. These findings advance the concept of psPEF as a bioelectronic tool for modulating proteostasis in neurodegenerative disease models.\u003c/p\u003e \u003cp\u003eOur data demonstrate that single-exposure psPEF treatment at 20 or 40 kV/cm reduces both the size and area of mHTT aggregates in HD-NSCs. This reduction was maintained at 24 hours post-treatment, suggesting durable reorganization or clearance of aggregate material. Importantly, the total number of aggregates per cell was largely unchanged, supporting a model in which psPEF primarily alters aggregate morphology or compactness rather than affecting seeding or transcription. This aligns with modeling studies suggesting that ultrashort electric fields can induce β-sheet to α-helix transitions in amyloidogenic proteins, destabilizing aggregate structures [74].\u003c/p\u003e \u003cp\u003eSupporting this mechanism, \u003cem\u003eHTT\u003c/em\u003e transcript levels remained unchanged following psPEF exposure, indicating that the disaggregation effect was not attributable to reduced expression. Instead, the effects appear to occur at the post-translational level. The dose-responsiveness of this effect\u0026mdash;where more robust changes occurred at 40 kV/cm\u0026mdash;further supports a field-strength dependent biophysical modulation of protein structure or intracellular trafficking. It remains to be determined whether the observed reduction in aggregate burden reflects true disaggregation, intracellular redistribution, or degradation via proteasomal or autophagic clearance. Future studies incorporating proteostasis pathway reporters and selective inhibitors will be critical to resolve these possibilities.\u003c/p\u003e \u003cp\u003eInterestingly, the spatial distribution of aggregates was differentially affected by psPEF. Cytosolic aggregates exhibited the most pronounced reductions, whereas nuclear aggregates transiently increased at 30 minutes before returning to baseline. One hypothesis is that larger aggregates may fragment and redistribute into nuclear compartments where clearance is slower or more compartmentalized. Alternatively, psPEF may differentially affect nuclear envelope permeability or aggregate trafficking mechanisms. While speculative, these results underscore the importance of subcellular localization in determining psPEF responses.\u003c/p\u003e \u003cp\u003eOur primary aim was to assess the modulation of existing aggregate burden within HD-derived cells. This within-line design enables interpretation of treatment effects without confounding by interline variability. Future studies incorporating isogenic WT-NSCs or time-course recovery analysis could further validate the specificity and longevity of the observed disaggregation effects. While neurons are the ultimate target of HD pathology, NSCs offer a biologically relevant and experimentally tractable model system for probing early aggregation phenotypes [54\u0026ndash;57]. Their preserved stress response pathways, rapid expansion, and lineage consistency make them well suited for high-content, mechanistically focused investigations of intracellular remodeling [75]. Prior studies have used NSCs to model protein aggregation dynamics and validate modulatory interventions before translation to mature neuronal systems [53,57,58].\u003c/p\u003e \u003cp\u003eIn addition to structural changes, we observed downregulation of two genes, \u003cem\u003ePAX6\u003c/em\u003e and \u003cem\u003eCACNA1C\u003c/em\u003e, following psPEF treatment. While these targets were not initially hypothesized, their modulation raises interesting possibilities. \u003cem\u003ePAX6\u003c/em\u003e is a neurodevelopmental transcription factor associated with progenitor maintenance and identity, and its reduction may suggest a shift toward neuronal maturation or stress adaptation [76]. \u003cem\u003eCACNA1C\u003c/em\u003e, encoding the Cav1.2 L-type calcium channel, regulates calcium influx and synaptic plasticity and is dysregulated in several neuropsychiatric conditions. Suppression of \u003cem\u003eCACNA1C\u003c/em\u003e could reflect altered excitability or intracellular calcium homeostasis\u0026mdash;both processes influenced in HD pathology [77,17]. The precise relationship between psPEF exposure, transcriptional regulation, and protein aggregate dynamics remains to be fully elucidated.\u003c/p\u003e \u003cp\u003eWe also observed a trend toward increased ΔΨm as assessed by TMRE staining, most notably at the higher field strength \u0026mdash;which is intriguing given that HD cells typically start from a chronically depolarized baseline [69\u0026ndash;71]. While these results did not reach statistical significance, possibly due to sample variability and temporal resolution limitations, the directionality aligns with reports that aggregate reduction may restore mitochondrial polarization in HD models [78]. Two, not mutually exclusive, mechanisms could make such a transient hyperpolarization feasible. First, modelling studies predict that each sub‑nanosecond pulse generates opposite charge accumulations across the inner mitochondrial membrane, producing momentary anode‑facing hyperpolarization before the net depolarizing phase dominates; integrating many pulses at 1 kHz could therefore bias the average ΔΨm upward in the short term [47,30,79]. Second, nsPEF exposure at sub‑lethal doses has been shown to stimulate electron‑transport flux and reduce proton leak, thereby increasing ΔΨm in other cell types\u0026mdash;a hormetic response later confirmed in HD neurons when PINK1 activation restored ΔΨm and viability [80\u0026ndash;83]. Live-cell potentiometric assays like TMRE are inherently variable due to mitochondrial network heterogeneity and cell-to-cell differences in dye loading. The modest sample size and early time-point focus may have limited statistical power despite biologically plausible trends. The 30-minute window was chosen to capture acute, primary responses in mitochondrial potential that may reflect direct effects of psPEF. Although post-hoc TMRE staining at 24 hours could theoretically provide insight into longer-term adaptations, we elected not to perform this due to both technical and biological limitations. Delayed TMRE application introduces greater susceptibility to dye-loading artifacts and reduced dynamic range, particularly in cells with altered metabolic states. Moreover, mitochondrial remodeling over extended intervals in HD models can obscure primary psPEF effects, making early time points more reliable for mechanistic interpretation. Future use of genetically encoded voltage indicators may allow improved resolution of these dynamics.\u003c/p\u003e \u003cp\u003eMechanistically, one potential pathway linking psPEF exposure to these effects involves intracellular calcium signaling. Several studies have reported that subnanosecond electric fields can induce calcium influx through voltage-gated calcium channels (VGCCs), particularly in cells that endogenously express these channels [74]. In our model, VGCC-mediated activation is plausible and consistent with prior findings that psPEF induces calcium transients without gross electroporation. However, we acknowledge that VGCC gating typically operates on microsecond timescales, which far exceeds the duration of our stimulus. Thus, conventional depolarization is unlikely to fully explain the observed effects. Instead, we propose that dielectric mechanisms\u0026mdash;such as local field-induced protein conformational changes or water dipole reorientation\u0026mdash;may contribute to transient activation or sensitization of these channels [37,84].\u003c/p\u003e \u003cp\u003eIn parallel with potential ion channel effects, we also propose that psPEF exposure may act directly on misfolded proteins or organelles via field-induced biophysical mechanisms. Given the subnanosecond rise time and field strengths applied here (20\u0026ndash;40 kV/cm), psPEF may transiently disrupt stabilizing interactions within polyglutamine aggregates through dipolar polarization or electroconformational stress. This hypothesis is supported by molecular dynamics simulations showing that ultrashort electric fields can induce β-sheet to α-helix transitions in amyloidogenic proteins, destabilizing fibrillar structures without the need for chemical chaperones [74,49]. Additionally, psPEF may influence cytoskeletal transport or cytoplasmic viscosity, facilitating the redistribution of aggregates observed post-treatment. Though membrane nanoporation is unlikely under these conditions, picosecond pulses have been shown to affect organelle membrane potential and protein function through dielectric polarization and water dipole reorientation [84,60,85,61]. These complementary mechanisms may help explain the observed changes in aggregate morphology and mitochondrial behavior while maintaining cell viability.\u003c/p\u003e \u003cp\u003eAlternatively, nanoporation cannot be entirely ruled out, particularly at higher field strengths. Although subnanosecond pulses are typically below the membrane charging time constant, molecular dynamics simulations suggest that extreme field strengths can induce water finger formation and transient nanopores within picoseconds under specific conditions [86]. It remains possible that some degree of non-lethal nanoporation or indirect ion channel modulation occurs, particularly in more exposed cytosolic regions. Therefore, we interpret VGCC activation as a likely but not exclusive pathway, and future studies with channel blockers, high-speed imaging, or electrophysiological recordings will be required to delineate these mechanisms more definitively.\u003c/p\u003e \u003cp\u003eFurther, while prior research using nsPEFs often emphasizes apoptosis induction or irreversible electroporation for cancer ablation, our study deliberately applies lower field strengths to avoid cytotoxicity. Indeed, we observe no loss in viability across conditions, supporting the concept that psPEF can modulate intracellular targets non-destructively. This distinction underscores the versatility of psPEF as a biophysical tool: unlike nsPEF-driven apoptosis or membrane rupture, psPEF may enable intracellular remodeling with minimal off-target damage when appropriately tuned [78,74].\u003c/p\u003e \u003cp\u003eWhile our study focused on early and intermediate responses, the 24-hour window was intentionally chosen to capture mechanistic changes while ensuring experimental consistency. Longer-term studies will be needed to determine whether psPEF-induced disaggregation leads to functional recovery, neuronal differentiation, or downstream clearance pathways. However, given the urgency of advancing psPEF as a novel modality, this window was appropriate for delineating short-term cellular responses. While no significant temperature rise was observed in earlier studies using similar conditions (e.g., via pH-sensitive dye methods), future iterations of the system will incorporate real-time thermal imaging or thermocouple feedback to confirm the absence of joule heating. Although simulation geometries were simplified and did not include the dielectric properties of well plate materials or fluid layers, ongoing refinements aim to improve the accuracy of modeled field distributions.\u003c/p\u003e \u003cp\u003eLooking forward, the non-contact nature of psPEF presents a critical advantage for translational application. Unlike traditional electrode-based systems that require direct physical contact, future implementations could leverage dielectric-loaded wideband antennas to deliver focused picosecond pulses to targeted tissues at depth. Recent simulations and in situ measurements demonstrate that such antennas can transmit high-intensity fields (\u0026ge;\u0026thinsp;20 kV/cm) several centimeters into subcutaneous targets, including the brain, with spatial precision in the sub-centimeter range [61]. This proof-of-concept antenna design addresses key engineering challenges in minimizing reflection losses at tissue-air interfaces and maintaining pulse fidelity. When combined with the non-lethal, intracellularly-targeted effects demonstrated in our study, these advances support a broader vision for psPEF as a contactless therapeutic platform for modulating proteostasis in vivo.\u003c/p\u003e \u003cp\u003eIn summary, our findings demonstrate that picosecond pulsed electric fields can non-lethally remodel polyglutamine aggregates in human HD neural stem cells, inducing durable changes in aggregate morphology and localization without compromising cell viability. While the precise molecular pathways remain to be elucidated, our data suggest that psPEF exerts its effects through a combination of field-induced biophysical mechanisms and transcriptional modulation. The lack of HTT transcript change, coupled with morphological disaggregation and redistribution, points to post-translational processes as a key component of the response. Importantly, this study provides proof-of-concept that psPEF can be leveraged as a contactless modality to interrogate and modulate proteostasis within intact human cells. These findings lay the groundwork for future exploration of psPEF as a therapeutic or investigative tool across a broad spectrum of aggregation-driven neurodegenerative diseases. While the present study focused on morphological and molecular changes in aggregate burden, future studies will be needed to determine whether psPEF-induced disaggregation translates into functional recovery. Assessments of synaptic signaling, differentiation status, and calcium dynamics will be essential to establish therapeutic relevance in mature neuronal systems.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was funded by institutional start-up support from the Ellmer College of Health Sciences at Old Dominion University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e M.T. performed the experiments, conducted data analysis, and contributed to manuscript preparation. M.Z., E.O., A.U.A., and C.K. assisted with experimental procedures. T.R.C. contributed to the statistical analysis of the data. R.A.P. provided technical expertise related to pulse delivery parameters. P.A.M., P.C.S., and R.D.B. jointly conceived and designed the study, supervised the research, interpreted the findings, and critically edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Number:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterial Transfer Agreement:\u003c/strong\u003e The Huntington’s disease neural stem cell lines and associated imaging data can be provided by Old Dominion University pending scientific review and a completed material transfer agreement. Requests for these materials should be submitted to: Peter A. 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Bioelectromagnetics 22 (6):440-448. doi:10.1002/bem.71\u003c/li\u003e\n\u003cli\u003eIbrahimi N, Vallet L, Andre FM, Ariztia L, Rivaletto M, de Ferron AS, Novac BM, Mir LM, P\u0026eacute;castaing L (2020) A Subnanosecond Pulsed Electric Field System for Studying Cells Electropermeabilization. Ieee T Plasma Sci 48 (12):4242-4249. doi:10.1109/Tps.2020.3034286\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"picosecond pulse electric fields (psPEF), protein aggregation, Huntington’s disease, bioelectric modulation, neural stem cells (NSCs), mitochondrial membrane potential (ΔΨm)","lastPublishedDoi":"10.21203/rs.3.rs-6486690/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6486690/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuntington\u0026rsquo;s disease (HD) is a neurodegenerative disorder marked by polyglutamine (PolyQ) aggregation and mitochondrial dysfunction, yet non-invasive methods to modulate these intracellular stressors remain limited. Here, we applied picosecond pulsed electric fields (psPEF)\u0026mdash;an ultrashort bioelectronic modality\u0026mdash;to patient-derived HD neural stem cells (NSCs) to assess changes in pathogenic protein aggregation, gene expression, and mitochondrial membrane potential. Using a custom 3D bioprinter-based stimulation platform, cells were exposed to non-contact electric fields of 20 or 40 kV/cm with subnanosecond pulse width (660 ps). Quantitative imaging and automated analysis revealed a significant reduction in aggregate size and aggresome burden within 30 minutes post-treatment, effects that persisted at 24 hours without compromising viability. HTT mRNA levels remained unchanged, supporting a post-translational mechanism of aggregate modulation. We also observed a transient redistribution of aggregates into the nuclear compartment and a field-dependent trend toward increased mitochondrial polarization, suggestive of broader proteostatic or bioenergetic effects. Transcript analysis revealed downregulation of PAX6 and CACNA1C, further implicating psPEF in modulating intracellular stress pathways. These findings represent the first evidence that ultrashort electric fields can reduce mutant HTT aggregation in a human HD model without genetic manipulation or membrane poration. Our results establish HD-NSCs as a scalable, disease-relevant platform for evaluating psPEF in neurodegenerative disease and support further exploration of dielectric mechanisms for intracellular remodeling. Collectively, this work introduces a contactless, non-invasive strategy for modulating protein aggregation and mitochondrial stress in human neural cells, offering a new direction for therapeutic development in proteopathy-driven conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Picosecond Pulsed Electric Field-Induced Disaggregation of Polyglutamine Aggregates in Huntington’s Disease Neural Stem Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-21 08:43:18","doi":"10.21203/rs.3.rs-6486690/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7227f25c-0c81-4ce7-92ac-52ee3436a959","owner":[],"postedDate":"May 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-30T14:43:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-21 08:43:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6486690","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6486690","identity":"rs-6486690","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}