Degron peptide targeting Ataxin-2 mitigates neurodegeneration and neuroinflammation Progression in a TDP-43 Mouse Model of ALS

Degron peptide targeting Ataxin-2 mitigates neurodegeneration and neuroinflammation Progression in a TDP-43 Mouse Model of ALS | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 18 June 2025 V1 Latest version Share on Degron peptide targeting Ataxin-2 mitigates neurodegeneration and neuroinflammation Progression in a TDP-43 Mouse Model of ALS Authors : Jingyan Zhu , Lixia Wang , Wendy Wen , Ljubomir Kojic , and Max Cynader [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175023624.41125652/v1 372 views 272 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease marked by progressive degeneration of upper and lower motor neurons, accompanied by neuroinflammation and TDP-43 proteinopathy, leading to muscle atrophy and paralysis. Ataxin-2 has been identified as a key modulator of TDP-43 toxicity, and its reduction has been shown to alleviate neurodegeneration and improve survival in ALS models, making it a promising therapeutic target for modifying disease progression. In this study, we developed lipid-modified degron peptides targeting Ataxin-2 for proteasomal degradation. We performed high-density peptide screening followed by 3D structure modeling to identify the optimal Ataxin-2 binding sequence. In vitro experiments demonstrated that degron peptides induce dose- and time-dependent degradation of Ataxin-2 in primary cultured neurons. To enhance peptide stability and tissue penetration, we employed lipidation strategies incorporating C20 and C16 fatty acid modifications, which significantly improved degron peptide efficacy in vivo. In TAR4/4 ALS mice, lipid-modified Ataxin-2-targeting degron peptides ameliorated motor neuron loss, improved motor function, and prolonged survival. Interestingly, despite these therapeutic benefits, TDP-43 aggregation was not significantly reduced, suggesting that Ataxin-2 depletion exerts effects through mechanisms beyond direct TDP-43 modulation. However, our findings showed a significant reduction in neuroinflammation in TAR4/4 ALS mice following peptide treatment. These results not only establish lipid-modified degron peptides as a viable therapeutic strategy for ALS but also provide a broader framework for targeting disease-relevant proteins implicated in neurodegeneration. This study paves the way for developing precision-targeted therapeutics with enhanced stability, bioavailability, and efficacy for ALS and other neurodegenerative diseases. Introduction Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that primarily affects motor neurons in the brain and spinal cord, leading to muscle weakness, paralysis, and eventual respiratory failure and death [1, 2]. Despite its complexity, the aggregation and mislocalization of TDP-43, a DNA/RNA-binding protein, have emerged as critical hallmarks of ALS pathology, with TDP-43 inclusions identified in over 97% of sporadic ALS cases [3-5]. The toxic gain of function associated with TDP-43 aggregation and the consequent loss of nuclear function contribute to motor neuron degeneration [6]. However, therapeutic strategies targeting TDP-43 directly have been challenging due to its essential cellular functions [7]. A potential solution lies in targeting modulators of TDP-43 toxicity, such as Ataxin-2 [8]. Ataxin-2 is a polyglutamine-containing protein involved in RNA metabolism, including mRNA stability, translation, and stress granule dynamics [9]. Genetic studies have identified Ataxin-2 variation as a risk factor for ALS, with intermediate-length polyglutamine expansions increasing susceptibility to the disease [10]. Importantly, recent work indicates that Ataxin-2 interacts with TDP-43 and regulates its function and toxicity [11]. Genetic Reduction of Ataxin-2 levels has been shown to suppress TDP-43 aggregation, ameliorate neurodegeneration, and extend survival in preclinical ALS models, making Ataxin-2 a compelling therapeutic target [12-14]. Understanding the mechanisms by which Ataxin-2 influences TDP-43 aggregation and toxicity is critical for the development of targeted therapies. One promising approach for targeting Ataxin-2 is the use of peptide degrons, short peptide sequences that bind specific proteins and direct them to the proteasome for degradation [15]. By hijacking the ubiquitin-proteasome system, degron peptides can selectively reduce the levels of pathogenic proteins, offering a novel therapeutic strategy for proteinopathies such as ALS [16]. This study explores the development of Ataxin-2-targeted degron peptides to evaluate their efficacy in reducing Ataxin-2 expression and, consequently, TDP-43 pathology in ALS models. However, one of the major challenges in peptide-based therapeutics is the short half-life of peptides in circulation, which limits their bioavailability and therapeutic efficacy. To overcome this limitation, we employed peptide lipidation, a chemical modification that involves attaching fatty acid chains, such as C20 or C16 lipids, to the peptide structure. Lipidation has been shown to enhance the pharmacokinetic properties of peptides by increasing their half-life in the bloodstream through binding to human serum albumin (HSA), which improves stability and resistance to enzymatic degradation [17]. Additionally, lipidation facilitates cellular uptake, further enhancing peptide efficacy [18]. By integrating lipidation into the design of Ataxin-2-targeted degron peptides, we aimed to develop a more stable, bioavailable, and effective therapeutic capable of mitigating ALS pathology. In this study, we systematically evaluated the therapeutic potential of lipid-modified Ataxin-2-targeted degron peptides in TAR4/4 mice, a transgenic model of ALS characterized by TDP-43 pathology and motor neuron degeneration [19]. We investigated the ability of these peptides to reduce Ataxin-2 expression, decrease TDP-43 aggregation, and ameliorate neuroinflammation, including microglial activation and astrocyte reactivity, which are prominent contributors to ALS progression. Furthermore, we assessed motor neuron survival and overall motor function to determine the therapeutic efficacy of these peptides. By combining the selective degradation capability of degron peptides with the enhanced stability provided by lipidation, this study represents a significant step toward the development of next-generation peptide therapeutics for ALS and other TDP-43 proteinopathies. Identification and Characterization of Ataxin-2-Targeted Degron Peptides To identify peptides capable of targeting Ataxin-2 for proteasome-mediated degradation, we performed systematic peptide screening using a high-density peptide array, followed by validation in in vitro models (Figure 1A). Candidate peptides were derived from reported Ataxin-2 protein binding partners and encoded onto the array for screening against the Ataxin-2 protein (Figure 1B). Two candidate peptides, originating from DDX6 and A2BP1 proteins, exhibited high binding affinity to Ataxin-2, and their structural positions were mapped onto the tertiary structure of the corresponding proteins (Figure 1C). Candidate peptide A was localized within the protein-binding pocket, indicating a potential role in authentic protein-protein interactions. In contrast, Candidate Peptide B was embedded in the internal protein structure, suggesting potential nonspecific binding due to its linearized structure on the array (Supplemental video 1 and 2). Using these candidates, degron peptides were subsequently engineered by conjugating signal peptides at the C-terminus to induce proteasomal degradation, along with a TAT sequence at the N-terminus to enhance cellular uptake. The efficacy of the degron peptides was evaluated in primary cultured neurons, focusing on dose-responses and time-dependent degradation. Western blot analysis revealed a concentration-dependent reduction in Ataxin-2 levels, with significant degradation observed from 1 μM to 50 μM (Figure 1D). Furthermore, time-course studies demonstrated significant decreases in Ataxin-2 expression as early as 3 hours post-treatment, with maximal degradation observed at 24 hours (Figure 1E). Collectively, these findings demonstrate that the degron peptides effectively bind and degrade Ataxin-2 in a dose- and time-dependent manner, providing a foundation for further evaluation in vivo. Fatty Acid Modification Enhances Peptide Efficacy for Ataxin-2 Degradation To establish a baseline for efficacy, we first evaluated the unmodified degron peptides in vivo. Western blot analysis of brain tissue from C57BL/6J mice treated with 60 mg/kg of the native peptide revealed a 54.67 ± 4.63% reduction in Ataxin-2 protein levels. However, the overall extent and duration of degradation were limited, with maximal suppression observed at 6 hours post-administration followed by a rebound in protein levels, suggesting suboptimal in vivo stability and/or cellular uptake of the native peptide (Supplemental Figure 1). To address these limitations, we engineered lipid-modified degron peptides by conjugating long-chain fatty acids—either a C20 dicarboxylic acid (Figure 2A) or a C16 monocarboxylic acid (Figure 2F)—to the peptide backbone. These chemical modifications were designed to enhance peptide pharmacokinetic properties, including plasma half-life and tissue penetration. We then assessed the dose-response and time-dependent efficacy of lipid-modified peptides in degrading Ataxin-2 in brain and spinal cord tissues in C57 mice. For C20-modified peptides, Western blot analysis revealed a dose-dependent reduction in Ataxin-2 expression with increasing peptide concentrations ranging from 1 to 60 mg/kg (Figure 2B and 2C). Maximal reductions in Ataxin-2 levels were observed at 10 mg/kg in brain tissue and 20 mg/kg in spinal cord tissue. Time-course analysis showed that C20-modified peptides significantly decreased Ataxin-2 levels as early as 3 hours post-treatment, with maximal degradation achieved at 24 hours in the spinal cord and 48 hours in the brain (Figure 2D and 2E). Notably, Ataxin-2 levels remained suppressed for up to 96 hours in the spinal cord, demonstrating the prolonged effects of C20-modified peptides. Similarly, C16-modified peptides exhibited dose-dependent efficacy in reducing Ataxin-2 levels across both tissues, with significant effects observed as low as 0.5 mg/kg in the brain and 1mg/kg in the spinal cord (Figure 2G and 2H). Time-course analysis revealed significant Ataxin-2 degradation starting at 3 hours post-treatment, with maximal suppression at 24 hours in the brain and spinal cord (Figure 2I and 2J). However, the suppression of Ataxin-2 levels began to wane after 72 hours, suggesting a shorter duration of action compared to C20-modified peptides. Overall, these findings demonstrate that both C20- and C16-modified peptides effectively degrade Ataxin-2 in a dose- and time-dependent manner. Notably, C20-modified peptides exhibited sustained suppression of Ataxin-2 over longer durations, whereas C16-modified peptides achieved greater reductions at lower doses. These results highlight the therapeutic potential of fatty acid-modified degron peptides with enhancing both peptide stability and effectiveness. Lipid-Modified Peptides Targeting Ataxin-2 Degradation Improve Survival and Motor Function in TAR4/4 Mice We evaluated the therapeutic potential of lipid-modified degron peptides targeting Ataxin-2 degradation in TAR4/4 mice, a well- established transgenic model of ALS. Treatment began at postnatal day 14, coinciding with the onset of motor dysfunction in most TAR4/4 mice. Mice were administered either C20-modified peptide (20 mg/kg, subcutaneously every other day) or C16-modified peptide (1 mg/kg, subcutaneously daily), while saline-treated mice served as controls (Figure 3A). Survival analysis demonstrated that both C20- and C16-modified peptides significantly extended the lifespan of TAR4/4 mice compared to saline-treated controls (Figure 3B). Although no statistically significant differences were observed between the C20- and C16-treated groups, it is noteworthy that one mouse in the C16-treated group exhibited an extended lifespan of up to 110 days. These findings suggest that both lipid-modified peptides confer a survival benefit in TAR4/4 mice, with some variability in individual responses to treatment. In addition, body weight was monitored throughout the study as an indicator of general health. Both C20- and C16-modified peptides significantly mitigated the progressive weight loss observed in saline-treated TAR4/4 mice, effectively preserving body weight during the treatment period (Figure 3C). Motor function was evaluated using several behavioral tests, including hind limb reflex, gait impairment, tremor, and kyphosis assessments. Treatment with both lipid-modified peptides significantly improved hind limb reflex scores, tremor, and kyphosis scores starting from postnatal day 16, and gait impairment starting from postnatal day 18, compared with saline-treated controls. These results indicated the therapeutic efficacy of lipid-modified peptides in alleviating motor deficits. In summary, the in vivo data demonstrate that lipid-modified degron peptides targeting Ataxin-2 degradation improve survival, reduce weight loss, and enhance motor function in TAR4/4 mice. The sustained efficacy and superior therapeutic effects observed with lipid-modified peptides underscore their potential as a robust strategy for mitigating ALS-related deficits through Ataxin-2 degradation. not-yet-known not-yet-known not-yet-known unknown Lipid-Modified Peptides Targeting Ataxin-2 Degradation Preserve Motor Neurons in TAR4/4 Mice To assess the neuroprotective effects of lipid-modified degron peptides, we quantified motor neuron survival in the primary motor cortex (M1) and lumbar spinal cord (L3–L5) of P26 TAR4/4 mice treated with C20- or C16-modified peptides. In the primary motor cortex (M1), NeuN immunostaining revealed significant motor neuron loss in saline-treated TAR4/4 mice compared to nTg controls. Treatment with C20- or C16-modified peptides significantly improved motor neuron density compared to saline-treated TAR4/4 mice (Figure 4A). Similarly, ChAT immunostaining of lumbar spinal cord segments (L3–L5) showed substantial motor neuron loss in saline-treated TAR4/4 mice compared to nTg controls. Both C20- and C16-modified peptides significantly preserved motor neurons in the ventral horn of L3, L4, and L5 spinal cord segments compared to saline-treated TAR4/4 mice (Figure 4B). These results indicate that lipid-modified peptides targeting Ataxin-2 degradation effectively preserve motor neurons in the brain and spinal cord of TAR4/4 mice, supporting their potential as therapeutic candidates for treating ALS-related motor neuron degeneration. Lipid-Modified Peptides Targeting Ataxin-2 Do Not Significantly Reduce TDP-43 Aggregation in TAR4/4 Mice To investigate whether lipid-modified degron peptides targeting Ataxin-2 degradation impact TDP-43 aggregation, we analyzed brain and spinal cord tissues from P21 TAR4/4 mice treated with C20- or C16-modified peptides. Western blot analysis quantified insoluble TDP-43 and phospho-TDP-43 (409/410) levels in both tissues. In the brain, insoluble TDP-43 levels were significantly elevated in saline-treated TAR4/4 mice compared to nTg controls (Figure 5A). Neither C20- nor C16-modified peptides significantly reduced insoluble TDP-43 levels (Figure 5B). Phospho-TDP-43 (409/410) was not detectable in either the insoluble or soluble fraction. The ratio of 35 kDa TDP-43 fragments in the soluble fraction was increased in saline-treated TAR4/4 mice compared to nTg controls but showed no significant difference between peptide-treated and saline-treated groups (Figure 5C). However, Ataxin-2 levels were significantly reduced in both peptide-treated groups compared to saline-treated TAR4/4 mice, confirming successful Ataxin-2 degradation (Figure 5D). In the spinal cord, insoluble TDP-43 levels were similarly elevated in saline-treated TAR4/4 mice compared to nTg controls (Figure 5E). C20- and C16-modified peptides did not significantly alter the levels of insoluble TDP-43 (Figure 5F) or the ratio of 35 kDa TDP-43 fragments in the soluble fraction (Figure 5G). Consistent with brain tissue, Ataxin-2 levels were markedly reduced in the spinal cords of peptide-treated groups, demonstrating the efficacy of the peptides in degrading Ataxin-2 (Figure 5H). These results indicate that while lipid-modified peptides effectively improve survival and motor function in TAR4/4 mice, they do not mitigate TDP-43 aggregation, suggesting alternative mechanisms of therapeutic benefit. not-yet-known not-yet-known not-yet-known unknown Lipid-Modified Peptides Targeting Ataxin-2 Reduce Microglial Activation in TAR4/4 Mice Neuroinflammation, driven primarily by activated microglia, plays a critical role in ALS pathogenesis, contributing to motor neuron degeneration and disease progression [20]. While Ataxin-2 is primarily known for its role in regulating TDP-43 toxicity, emerging evidence suggests that modulating Ataxin-2 levels may also influence inflammatory pathways [21]. Given the growing recognition of neuroinflammation as a major contributor to ALS pathology, we sought to determine whether lipid-modified peptides targeting Ataxin-2 degradation could mitigate microglial activation, thereby addressing another key aspect of disease pathology. To assess this, we analyzed microglial activation in the cortex and lumbar spinal cord of P21 TAR4/4 mice using Iba-1 immunostaining, serving as a marker of inflammation. In the cortex, microglial activation was significantly elevated in saline-treated TAR4/4 mice compared to nTg controls, as indicated by increased Iba-1+ cell density and fluorescence intensity. Treatment with both C20- and C16-modified peptides significantly reduced Iba-1+ cell density compared to saline-treated TAR4/4 mice (Figure 6A). In the lumbar spinal cord, a similar trend was observed. Saline-treated TAR4/4 mice exhibited significantly higher microglial activation compared to nTg controls, reflected by increased Iba-1+ cell density and fluorescence intensity. Treatment with C20- and C16-modified peptides significantly decreased microglial activation, with a pronounced reduction in Iba-1+ cell density (Figure 6B). These results demonstrate that lipid-modified peptides targeting Ataxin-2 effectively reduce microglial activation in both the brain and spinal cord of TAR4/4 mice. The superior efficacy of the lipid-modified peptides further highlights their potential as a therapeutic approach for mitigating neuroinflammation in ALS-related models. Lipid-Modified Peptides Targeting Ataxin-2 Reduce Astrocyte Activation in TAR4/4 Mice Astrocyte activation is another hallmark of neuroinflammation that is thought to contribute to disease progression in ALS by amplifying inflammatory signals and exacerbating neuronal damage [22]. To evaluate the effects of lipid-modified peptides targeting Ataxin-2 degradation on astrocyte activation, we analyzed GFAP immunostaining in the cortex and lumbar spinal cord of P21 TAR4/4 mice. In the cortex, GFAP immunostaining revealed a significant increase in astrocyte activation in saline-treated TAR4/4 mice compared to nTg controls, particularly in cortical layer II/III and V. Treatment with both C20- and C16-modified peptides significantly reduced the GFAP+ signals in TAR4/4 mice compared to saline controls, (Figure 7A). A similar pattern was observed in the lumbar spinal cord, where GFAP+ immunostaining was markedly elevated in saline-treated TAR4/4 mice compared to nTg controls, indicating robust astrocyte activation. Both C20- and C16-modified peptides significantly decreased astrocyte activation in the spinal cord (Figure 7B). These results demonstrate that lipid-modified peptides targeting Ataxin-2 effectively reduce astrocyte activation in both the brain and spinal cord of TAR4/4 mice. The pronounced effect of lipid-modified peptides further supports their potential as therapeutic agents for mitigating neuroinflammation in ALS-related pathology. Discussion This study highlights the therapeutic potential of lipid-modified degron peptides targeting Ataxin-2 degradation in ALS. Ataxin-2 has been identified as a key modulator of TDP-43 toxicity, with intermediate-length polyglutamine (polyQ) expansions in Ataxin-2 associated with an increased risk of ALS and an enhancement of TDP-43 toxicity, as demonstrated by genetic studies in patient cohorts and disease models [23, 24, 25]. Previous studies have shown that genetic knockdown of Ataxin-2 ameliorates neurodegeneration and extends survival in ALS models [12-14]. Unlike traditional approaches such as genetic knockdown or RNA-based therapies, degron peptides provide unique advantages by directly targeting proteins for degradation. This approach enables more precise control over protein levels and allows for modulation of disease-relevant proteins without altering upstream DNA or RNA pathways, which often carry risks of off-target effects or incomplete silencing [26]. Additionally, antisense oligonucleotide therapies have been associated with immune activation, which may exacerbate neuroinflammation and contribute to treatment-related adverse effects [27]. By contrast, degron peptides may offer a safer alternative by avoiding the innate immune responses triggered by exogenous nucleic acids, potentially leading to better tolerability and therapeutic efficacy in ALS patients. In our initial in vitro experiments, unmodified degron peptides were tested in cultured neurons to evaluate their ability to degrade Ataxin-2. These peptides demonstrated dose- and time-dependent reductions in Ataxin-2 levels, with significant degradation observed at peptide concentrations as low as 1 µM and maximal reductions achieved at 24 hours post-treatment. Furthermore, Ataxin-2 degradation was detectable as early as 3 hours post-treatment, highlighting the fast-acting nature of degron peptides. Importantly, the proteasome-mediated degradation mechanism utilized by degron peptides closely aligns with the cell’s natural protein turnover processes, reducing the risk of toxic intermediate accumulation or degradation-resistant species. These properties, coupled with the observed low toxicity, underscore the safety and therapeutic potential of degron peptides as a novel approach for ALS treatment. One of the drawbacks of natural peptides is their rapid degradation and poor stability under physiological conditions, necessitating frequent administration in both in vivo animal studies and clinical applications. This limitation has historically hindered the broad adoption of peptide-based therapeutics. However, recent advancements in peptide engineering, such as lipidation, have significantly enhanced the pharmacokinetic properties of peptides. Lipidated peptides have demonstrated prolonged half-life, improved bioavailability, and enhanced tissue penetration, making them more effective in clinical settings. For instance, lipidation strategies have been successfully employed in approved therapeutics, such as liraglutide and semaglutide for diabetes, which utilize fatty acid modifications to achieve sustained drug action and reduced dosing frequency [28, 29]. These successes underscore the potential of lipidated peptides as a versatile platform for overcoming the inherent limitations of natural peptides, particularly in chronic conditions like ALS, where long-term and efficient therapeutic delivery is essential. In our study, we evaluated the dose-response and time-dependent efficacy of C20- and C16-modified peptides targeting Ataxin-2 degradation in brain and spinal cord tissues. Compared to their unmodified counterparts, both lipid-modified peptides exhibited markedly improved stability in the circulation and demonstrated efficacy at significantly lower doses. The C20-modified peptide showed the longest duration of action, with detectable effects persisting for up to 48 hours post-administration, while Ataxin-2 levels remained suppressed for up to 96 hours in the spinal cord. While the C16-modified peptide displayed significant stability for 24 hours, its efficacy was observed at doses as low as 0.5 mg/kg in the brain and 1mg/kg in the spinal cord, sharply reducing the effective in vivo dose. These enhancements reduced the need for frequent administration and allowed for consistent Ataxin-2 degradation in vivo. Furthermore, lipidation also improved tissue penetration, with robust delivery to both the brain and spinal cord, as evidenced by measurable peptide concentrations in target tissues. These results highlight the potential of lipidated peptides as a next-generation therapeutic modality for ALS by addressing the stability and delivery challenges of natural peptides. In this study, we demonstrated that degron peptides designed to degrade Ataxin-2 effectively mitigate several pathological hallmarks of ALS, including motor neuron loss, neuroinflammation, and motor deficits in TAR4/4 mice. However, they did not significantly reduce TDP-43 aggregation, suggesting that their therapeutic benefits arise through mechanisms beyond direct modulation of TDP-43 pathology. Ataxin-2 is known to influence TDP-43 through its roles in RNA metabolism, stress granule formation, and protein stability [9]. However, our data suggest that the observed therapeutic effects stem from indirect downstream pathways rather than a direct reduction of TDP-43 aggregation. A recent Drosophila study found that Ataxin-2 reduction partially restores RNA homeostasis disrupted by TDP-43-induced RNA instability and regulates transcripts enriched in small-molecule metabolic pathways [30], further supporting its role in modulating RNA metabolism. Additionally, crosslinking immunoprecipitation (CLIP) studies have revealed that both TDP-43 and Ataxin-2 bind to the same mRNA targets, suggesting that these RNA-binding proteins (RBPs) may co-regulate specific transcripts [31]. This highlights Ataxin-2’s critical role in RNA processing and metabolic regulation, which may contribute to the broader cellular dysfunction observed in TDP-43 pathology. Further supporting this, a study in iPSC-derived neurons showed that Ataxin-2 and TDP-43 belong to the same interactome [32]. Notably, ATXN2 knockdown alleviated TDP-43 overexpression-induced neuronal loss and reduced stress granule formation in iPSC-derived GABAergic neurons, reinforcing the idea that Ataxin-2 plays a critical role in modulating TDP-43 toxicity, RNA processing, and stress granule dynamics. These findings suggest that targeting Ataxin-2 could provide a therapeutic strategy for ALS by restoring RNA homeostasis, reducing neuronal stress, and mitigating TDP-43-driven cellular dysfunction. It is worth noting that our findings demonstrated that degron peptides targeting Ataxin-2 effectively reduce neuroinflammation in TAR4/4 mice, highlighting Ataxin-2 as a critical regulator of inflammatory responses in ALS. Increasing evidence suggests that TDP-43 toxicity contributes to neuroinflammation through multiple mechanisms, including innate immune activation, stress granule persistence, cytokine dysregulation, and inflammasome activation [33]. Our results indicate that Ataxin-2 degradation mitigates neuroinflammation through multiple downstream pathways, suggesting that Ataxin-2 either modulates TDP-43 toxicity by influencing its RNA metabolism and stress granule dynamics or functions as part of a broader inflammatory network independent of direct TDP-43 interactions. As we know, Ataxin-2 is a key regulator of stress granule (SG) formation and dynamics, and its interaction with TDP-43 contributes to SG persistence in ALS [32]. Persistent stress granules act as danger-associated molecular patterns (DAMPs), triggering innate immune responses through Toll-like receptors (TLRs) and type I interferon signaling, leading to sustained neuroinflammation [34]. However, we did not observe abnormal G3BP1 expression patterns in the spinal motor neurons of TAR4/4 mice, suggesting that the reduction in neuroinflammation following Ataxin-2 degradation is not mediated through SG dynamics. Interestingly, recent clinical efforts targeting protein homeostasis and the integrated stress response (ISR) in ALS have faced challenges. ABBV-CLS-7262, an EIF2B activator designed to counteract ISR-mediated shutdown of protein synthesis, recently failed in a phase 2 clinical trial despite preclinical evidence supporting its neuroprotective effects. This highlights the complexity of targeting stress response pathways in ALS and suggests that modulating protein homeostasis alone may not be sufficient to mitigate disease progression. In contrast, our approach targeting Ataxin-2 degradation may provide broader therapeutic benefits by modulating inflammatory signaling pathways more directly. To further elucidate the mechanisms by which Ataxin-2 depletion reduces neuroinflammation, we plan to perform RNA sequencing (RNA-seq) and CLIP-seq analysis on peptide-treated vs. untreated neurons to identify changes in the regulation of inflammatory transcripts. Additionally, ELISA or multiplex cytokine assays on spinal cord lysates and cerebrospinal fluid (CSF) can be conducted to quantify pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), validating the role of Ataxin-2 depletion in suppressing neuroinflammation through pathways independent of stress granule regulation. Given the emerging role of nuclear TDP-43 loss-of-function in ALS, cryptic exon formation has been identified as a key pathogenic mechanism. TDP-43 is essential for RNA splicing and transcript stability, and its nuclear depletion leads to the aberrant inclusion of cryptic exons, resulting in defective gene expression and neurotoxicity. This process disrupts essential neuronal functions, contributing to progressive degeneration in ALS [35, 36, 37]. Since Ataxin-2 regulates TDP-43-driven RNA metabolism, targeting Ataxin-2 for degradation may potentially increase the availability of nuclear TDP-43 for proper RNA splicing, help to restore RNA homeostasis, and reduce cryptic exon inclusion. Future studies will assess cryptic exon burden in TAR4/4 mice following Ataxin-2 degron peptide treatment, using RNA sequencing and splicing analysis to determine whether Ataxin-2 depletion provides neuroprotection by alleviating TDP-43 loss-of-function at the RNA level. Overall, this study demonstrates that degron peptides targeting Ataxin-2 degradation hold significant therapeutic promise for ALS by offering advantages over traditional genetic and RNA-based approaches through direct protein modulation. Lipid modification significantly enhances peptide stability, bioavailability, and efficacy, making these peptides more viable candidates for long-term therapeutic applications. Our findings suggest that Ataxin-2 depletion mitigates neurodegeneration and neuroinflammation. Future studies will focus on validating the impact of Ataxin-2 depletion on cryptic exon generation and inflammatory pathways, further clarifying its potential as a next-generation ALS therapeutic strategy. Methods Peptide array, degron peptide design, and peptide lipidation Peptide array The peptide spot array was synthesized on a cellulose-based membrane at the UBC Peptide Synthesis Facility using a previously published protocol [38]. The array contained overlapping 14-mer peptides with a two-amino acid shift per spot, covering the full sequence of DDX6, PABP1, and A2BP1 proteins. To initialize the membrane, it was washed twice with methanol for 10 minutes at room temperature, followed by three washes with TBST. Before bait protein binding, the membrane was blocked in TBST containing 5% sucrose and 4% non-fat dry milk for 4 hours at room temperature. It was then incubated overnight at 4°C with 10 µg/mL ATAXIN-2 recombinant protein in 4% non-fat dry milk or with 4% non-fat dry milk alone as a negative control. On the second day, the membrane was washed with TBST and incubated overnight at 4°C with a rabbit polyclonal ATAXIN-2 antibody. After washing in TBST, it was incubated with a secondary antibody for 2 hours at room temperature. Finally, the membrane was washed again in TBST and prepared for visualization of positive protein binding spots using an enhanced chemiluminescence assay. Degron peptide design and synthesis Degron peptides were designed based on sequence motifs known to mediate protein degradation through the ubiquitin-proteasome system. In this study, RRRG peptides were linked to the C-terminus to enhance degradation efficiency. Candidate degron sequences were identified using the peptide array, and a cell-penetrating TAT peptide was conjugated to the N-terminus to facilitate intracellular delivery. The selected degron sequences were synthesized using solid-phase peptide synthesis (SPPS) at the UBC Peptide Synthesis Facility. Peptides were purified by high-performance liquid chromatography (HPLC) and confirmed by mass spectrometry. Peptide lipidation The chosen Ataxin-2 degron peptide is a 28 amino acid linear peptide conjugated to either a C16 fatty acid or C20 fatty diacid moiety. To generate C16-modified peptides, we employed a short spacer linker, γGlu, connected to the peptide’s N-terminus. For C20 modification, a C20 fatty diacid was similarly attached via the spacer linker, γGlu-2xOEG, to the peptide’s N-terminus. The identity and purity (>95%) of the lipid–peptide conjugates were confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics). Additional verification of lipid incorporation was performed by electrospray ionization (ESI) mass spectrometry, which revealed mass shifts consistent with C16 or C20 moieties. Purified peptides were stored at –80°C until further use. Primary cortical neuron culture and peptide treatment Primary cortical neurons were isolated from E15 embryos of timed pregnant CD1 mice. Embryos were dissected in HBSS, and whole brains were isolated. The meninges were carefully removed, and the cortical lobes were separated and transferred to 15 mL tubes containing HBSS. After a gentle spin, the supernatant was replaced with 5 mL of 0.25% trypsin, and tissues were incubated at 37°C for 20 minutes for enzymatic digestion. The digested cortices were triturated using a 10 mL pipette and washed twice with DMEM containing 10% fetal bovine serum. Following centrifugation at 1500 rpm for 5 minutes, the cell pellet was resuspended in 5–10 mL neurobasal plating medium supplemented with 2% B27, 2 mM L-glutamine, 25 μM glutamic acid, 10 mM β-mercaptoethanol, and 1% penicillin-streptomycin. Cells were plated on poly-D-lysine-coated six-well plates at a density of 8 × 10⁵ cells per well. The medium was replaced on day 2, and half of the medium was refreshed every 3–4 days. Cultures were maintained at 37°C in a humidified incubator with 5% CO₂. Primary cortical neurons were cultured for 14 days in vitro (DIV) to allow for neuronal maturation prior to peptide treatment. Peptides were prepared as 10 mM stock solutions in double distilled water and subsequently diluted in Neurobasal medium to the desired working concentrations. Following the treatment, neurons were harvested for protein isolation, and Western blot analysis was performed to assess protein expression levels. Ethical Statement All animal procedures were approved by the University of British Columbia Animal Care Committee (ACC) and conducted in accordance with the Canadian Council on Animal Care (CCAC) guidelines. Animals All animal protocols were approved by the University of British Columbia Animal Care Committee (ACC). TDP-43 transgenic mice (TAR4 strain, B6; SJL-Tg (Thy1-TARDBP) 4Singh/J), originally generated by Samir Kumar-Singh, were purchased from The Jackson Laboratory (JAX, stock #012836). Mice were maintained on a B6/SJL background by crossing hemizygous mice with B6SJLF1/J F1 hybrids (stock #100012). Hemizygous mice were then intercrossed to produce TAR4/4 and non-transgenic offspring used in the experiments. All mice were housed in a temperature-controlled facility on a 12-hour light/dark cycle, with food and water provided ad libitum. Pups were weaned at postnatal day 21 (P21). Beginning at P12, litters received DietGel Boost High Calorie Dietary Supplement (ClearH 2 O, Westbrook, ME), placed on the cage floor to facilitate access for impaired mice. Genotyping Mice were individually identified by ear marks and tail-snipped at postnatal day 14 (P14) for genotyping. PCR was performed using the TDP-43 common primer 5’-TGAAATCCGGGTGGTATTGG-3’ (13790, JAX), the TDP-43 wildtype primer 5’-GGTGAGTTTAACCTTCAAGGGCT-3’ (13791, JAX), and the TDP-43 transgene primer 5’- AGCTTGCTAGCGGATCCAGAC-3’ (13792, JAX, Bar Harbor, ME) according to protocols recommended by The Jackson Laboratory (Bar Harbor, ME). Peptide injections Peptides were freshly prepared at 2 mg/mL in 0.9% saline immediately before administration. Subcutaneous (s.c.) injections were performed in the dorsal flank region using a 27-gauge needle. For dose-response and time-dependent experiments, a single injection was administered. In Tar 4/4 mice, the C16-modified degron peptides were injected daily, whereas the C20-modified degron peptides were administered every other day. Behavioral Analysis All measurements and scoring were performed as described [12, 19] and recorded from P14 until the endpoint. The following parameters were evaluated: The hindlimb reflex was scored on a scale of 3 to 0 based on hindlimb reflex extension: (3) normal hind limb position, (2) intermediate hind limb position, and (1) closed hind limb position. Gait impairment was assessed using a 0-4 scale, in which (0) the mouse walks normally, displaying no gait impairment; (1) the mouse exhibits a limp or tremor while walking; (2) the mouse has a severe limp, a severe tremor, a lowered pelvis, or outward-pointing feet (duck feet); (3) the mouse shows difficulty moving forward, minimal joint movement, an inability to use its feet for forward motion, difficulty remaining upright, or drags its abdomen on the ground; (4) the mouse has reached end-stage, falls over and cannot right itself within 30 seconds on three consecutive trials, prompting euthanasia. Tremor was evaluated on a 0-4 scale, in which (0) indicates no tremor; (1) indicates mild tremor while moving; (2) indicates severe tremor while moving; (3) indicates severe tremor both at rest and during movement; (4) indicates the mouse has reached end stage. Kyphosis was scored as follows: (0) the mouse can easily straighten its spine when walking and shows no persistent kyphosis; (1) mild curvature is present, but the mouse can still straighten its spine; (2) the mouse cannot fully straighten its spine and exhibits persistent but mild kyphosis; (3) pronounced kyphosis is maintained during walk or sitting; (4) the mouse has reached end stage. Body weight was recorded every other day, beginning at P14. For survival analysis, a humane euthanasia endpoint was employed, defined as a gait impairment score of 4, at which point mice were euthanized to minimize distress. Tissue collection and preparation TAR4/4 or non-transgenic mice were anesthetized, then perfused intracardially with ice-cold 0.9% saline, followed by ice-cold 4% paraformaldehyde (PFA) in PBS. Brains and spinal cords were dissected and fixed overnight in 4% PFA at 4°C. Tissues were then transferred to 30% sucrose in PBS for cryoprotection. After equilibration, they were embedded in O.C.T. compound and stored at –80°C. Tissue sections were subsequently prepared using a cryostat for immunofluorescence analysis. Immunohistochemistry Tissues were cryosectioned at a thickness of 30 µm using a Leica cryostat. Sections were washed twice in PBS, then blocked in 0.1% Triton X-100 and 5% BSA in PBS for 1 hour at room temperature. They were subsequently incubated overnight at 4°C with primary antibodies diluted 1:100 in 0.05% Triton X-100 and 3% BSA in PBS. On the second day, sections were washed three times in PBS and incubated with Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies (1:200) for 2 hours at room temperature. DAPI (1:500) was applied for 5 minutes to stain nuclei. Finally, sections were mounted onto glass slides and visualized using a confocal microscope. The following primary antibodies were used in this study: NeuN (Abcam ab177487, 1:500), ChAT (Abcam ab178850, 1:200), Iba-1 (Abcam ab225260, 1:200), GFAP (Thermo Fisher 13-0300, 1:100). Insoluble protein extraction Cells or tissues were harvested and lysed in RIPA buffer on ice. Lysates were sonicated and centrifuged at 20,000 × g for 15 minutes at 4°C, and the supernatant was collected as the soluble fraction. The remaining pellet was sonicated and washed three times in RIPA buffer, then resuspended in UREA buffer [7M Urea; 2M Thiourea; 2%SDS, 30mM Tris-HCl, pH 8.5 and protease inhibitor]. After sonication and a 40-minute incubation at room temperature, the mixture was centrifuged at 20,000 × g for 10 minutes at 4°C, and the supernatant was collected as the insoluble fraction. All protein samples were subsequently analyzed by Western immunoblotting. Western blotting Protein samples were mixed with 4× sample buffer and denatured at 100°C prior to SDS-PAGE. Notably, proteins suspended in UREA buffer were exempt from the denaturation step. Equal amounts of total protein were loaded into 5% stacking gels and resolved in 8% SDS–polyacrylamide resolving gels using a Bio-Rad electrophoresis system (Bio-Rad, Hercules, CA). Following electrophoresis, proteins were transferred onto PVDF membranes using a Bio-Rad Wet Transfer System at 100 V for 90 minutes at 4°C. Membranes were then blocked in 5% BSA prepared in TBST for 1 hour at room temperature, followed by overnight incubation at 4°C with primary antibodies. On the second day, membranes were washed in TBST and incubated for 1 hour with HRP-conjugated secondary antibodies (Goat anti-rabbit IgG, PerkinElmer NEF812001EA, 1:5000; or Goat anti-mouse IgG, PerkinElmer NEF822001EA, 1:5000). After another series of washes in TBST, signals were detected using an ECL substrate and visualized on a Bio-Rad ChemiDoc MP Imaging System. The relative protein density was quantified with Bio-Rad Quantity One software, normalized to actin on the same membrane. The following primary antibodies were used in this study: Ataxin-2 (Proteintech 21776-1-AP, 1:1000), actin (NEB 3700S, 1:1000), TDP-43 (Proteintech 10782-2-AP, 1:1000), phsosphoTDP-43 Ser409/410 (Cosmo CAC-TIP-PTD-M01, 1:1000). Image analysis All image processing and analysis were performed using ImageJ/Fiji. Motor neuron quantification was conducted on NeuN-positive neurons in motor cortex layer V and ChAT-positive neurons in the ventral horn of the lumbar spinal cord from immunostained coronal brain and spinal cord sections. Images were acquired using a confocal microscope under identical exposure and acquisition settings across all samples to ensure consistency. For each mouse, three to five non-overlapping fields of view per section were analyzed, with at least three sections per animal. For NeuN-positive neuron quantification, grayscale images were converted to binary, and a thresholding algorithm was applied to detect NeuN-positive signals. The Analyze Particles function in Fiji was used to count cells, with particle size (200~1500) and circularity parameters (0.4~1.0) optimized to exclude artifacts and background noise. Data were expressed as the average number of NeuN-positive neurons per 0.1mm². ChAT-positive cells were manually counted based on clear ChAT immunoreactivity and the presence of a distinct nucleus to avoid inclusion of fragmented or non-neuronal ChAT-expressing structures. Motor neuron density was expressed as the average number of ChAT-positive neurons per ventral horn per section. Microglial activation was assessed by Iba-1 immunostaining. For microglial density analysis, the Analyze Particles function in Fiji was used to quantify Iba-1-positive cells, with size (50–1500 µm²) and circularity (0.3–1.0) parameters optimized to exclude artifacts and background fluorescence. Data were expressed as the number of Iba-1-positive microglia per mm². Integrated density analysis was performed by measuring the total Iba-1 fluorescence intensity normalized to the total image area. To assess astrocyte activation, GFAP immunostaining was analyzed by measuring the percentage area covered by GFAP fluorescence in the spinal cord and motor cortex. Images were first converted to grayscale, and a thresholding algorithm was applied to detect GFAP-positive signal while minimizing background noise. The Measure Area function in Fiji was used to calculate the total GFAP-positive area as a percentage of the total image area. Identical thresholding settings were applied across all samples to ensure consistency. Statistical analysis Statistical analyses were conducted using GraphPad Prism. For comparisons between two groups, an unpaired two-tailed Student’s t-test was used. For comparisons among three or more groups, a one-way ANOVA followed by Tukey’s post hoc test was used. For time-course analyses, a two-way repeated-measures ANOVA with Bonferroni correction was performed to assess group differences over time. For survival analysis, Kaplan-Meier survival curves were generated, and statistical significance was determined using the log-rank (Mantel-Cox) test to compare survival distributions between groups. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was set at p < 0.05. Author contributions M.C. conceptualized the study and provided guidance throughout the project. J.Z. conducted all experiments, analyzed the data, and wrote the manuscript. L.K. prepared the animal protocol. L.W. synthesized and modified the peptides. 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Identification and Characterization of Ataxin-2-Targeted Degron Peptides (A) Schematic of the systematic peptide screening workflow, illustrating the process of identifying specific degron peptides for Ataxin-2 through high-density peptide array screening. (B) Peptide array epitope mapping reveals two candidate Ataxin-2 binding peptides derived from DDX6 and A2BP1 proteins. (C) Structural positions of each candidate peptide (shown as sticks) within the 3D tertiary structures of DDX6 and A2BP1 proteins, generated by the Swiss-Model. (D) Dose-response analysis of degron peptide efficacy in Ataxin-2 degradation. Western blots show Ataxin-2 and β-actin expression levels in primary cultured neurons at 4 hours post-treatment. (E) Time-dependent degradation of the Ataxin-2 protein in primary cultured neurons, as shown by Western blots following treatment with 10 μM degron peptide. Data are presented as mean ± SEM, n=3. *p < 0.05, **p < 0.01, ***p < 0.001. Figure 2. Western Blot Analysis for Dose-Response and Time-Dependent Effects of Lipid-Modified Ataxin-2-Targeted Degron Peptides in Brain and Spinal Cord in Mice (A) Schematic representation of the C20 fatty acid-modified Ataxin-2-targeted degron peptide structure. (B, C) Dose-response analysis of the C20-modified degron peptide’s efficacy in degrading Ataxin-2 in brain and spinal cord tissue after 4 hours of treatment in mice. (D, E) Time-dependent analysis of Ataxin-2 degradation in brain tissue treated with 20 mg/kg of the C20-modified degron peptide. (F) Schematic representation of the C16 fatty acid-modified Ataxin-2-targeted degron peptide structure. (G, H) Dose-response analysis of the C16-modified degron peptide’s efficacy in degrading Ataxin-2 in brain and spinal cord tissue after 4 hours of treatment in mice. (I, J) Time-dependent analysis of Ataxin-2 degradation in brain tissue treated with 1mg/kg of the C16-modified degron peptide. Data are presented as mean ± SEM, n=5, with statistical significance indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 3. Lipid-Modified Ataxin-2-Targeted Degron Peptides Improve Motor Function and Extend Survival of TAR4/4mice (A) Schematic of the experimental setup evaluating the efficacy of Ataxin-2-targeted degron peptides in TAR4/4 (TDP-43 Tg/Tg) mice. (B) Survival curve depicting the probability of survival over postnatal days for TAR4/4 mice treated with C20-modified peptide (20 mg/kg, subcutaneously every other day, n=11) and C16-modified peptide (1 mg/kg, subcutaneously daily, n=12), in comparison with the control group (saline, subcutaneously, n=25), Log rank test. (C) Body weight changes (%) over time in each group (n=12). (D-G) Behavioral assessments of motor function in TAR4/4 mice. Scores for hind limb reflex (D), gait impairment (E), tremor (F), and kyphosis (G) are shown for each treatment group (n=12), in comparison with the control group (n=12). Unpaired t test at each time point. Data are presented as mean ± SEM, with statistical significance indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 4. Neuroprotective Effects of Lipid-Modified Ataxin-2-Targeted Degron Peptides in TAR4/4 Mice (A) Representative images of NeuN-stained motor neurons in the primary motor cortex (M1) of P26 nTg and TAR4/4 (TDP-43 Tg/Tg) mice. Panels show comparisons between nTg saline (a), TAR4/4 saline (b), TAR4/4 mice treated with C20-modified peptide (20 mg/kg, subcutaneously every other day) (c), and TAR4/4 mice treated with C16-modified peptide (1 mg/kg, subcutaneously daily) (d). Enlarged images (a’-d’) highlight the area of interest indicated in (a-d). The right panel shows quantification of motor neuron counts in the primary motor cortex (M1) for each group (n=6). (B) Representative images of ChAT+ motor neurons in the ventral horn of the lumbar spinal cord (segments L3, L4, and L5) in P26 nTg and TAR4/4 mice. Panels show comparisons between nTg saline, TAR4/4 saline, TAR4/4 mice treated with C20-modified peptide (20 mg/kg, subcutaneously every other day), and TAR4/4 mice treated with C16-modified peptide (1 mg/kg, subcutaneously daily). The right panel shows quantification of ChAT+ motor neuron counts in lumbar spinal cord segments L3, L4, and L5 (n=6). Unpaired t test. Data are presented as means ± SEM, with statistical significance indicated as follows: *p < 0.05, ***p < 0.001, ****p < 0.0001, ###p < 0.001, ####p < 0.0001; ns = not significant. Error bar=100μm. Figure 5. Targeting Ataxin-2 Does Not Reduce TDP-43 Aggregation in TAR4/4 Mice (A) Representative Western blot images showing the expression of TDP-43, phosphorylated TDP-43 (Ser409/410), Ataxin-2, and β-actin in the insoluble and soluble fractions of brain tissues from P21 nTg and TAR4/4 (TDP-43 Tg/Tg) mice treated with saline, C20-modified peptide, or C16-modified peptide. (B-D) Quantification of the soluble/insoluble TDP-43 ratio (B), the soluble TDP-43 35 kDa fragment ratio (C), and the relative expression of Ataxin-2 (D), with data collected from three mice per group. (E) Representative Western blot images of TDP-43, phospho-TDP-43 (409/410), Ataxin-2, and β-actin expression in the insoluble and soluble fractions of Spinal Cord tissues from P21 nTg and TAR4/4 (TDP-43 Tg/Tg) mice treated with saline, C20-modified peptide, or C16-modified peptide. (F-H) Quantification of the soluble/insoluble TDP-43 ratio (F), the soluble TDP-43 35 kDa fragment ratio (G), and the relative expression of Ataxin-2 (H), with data collected from three mice per group. Unpaired t test. Data are presented as mean ± SEM, with statistical significance marked as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ##p < 0.01, ###p < 0.001; ns = not significant. Figure 6. Ataxin-2-Targeted Peptide Reduces Microglial Activation in TAR4/4 Mice Representative images of Iba-1 (green) immunostaining in the cortex (A) and lumbar spinal cord (B) of P21 nTg and TAR4/4 (TDP-43 Tg/Tg) mice. Panels show comparisons between nTg saline, TAR4/4 saline, TAR4/4 mice treated with C20-modified peptide, and TAR4/4 mice treated with C16-modified peptide. Merged images of Iba-1 (green) and DAPI (blue) demonstrate the localization and density of Iba-1+ microglial cells in the cortex and lumbar spinal cord in each group. Right panels show quantification of Iba-1+ cells per mm2 and Iba-1 fluorescence intensity for each group. Unpaired t test. Data are presented as mean ± SEM, with statistical significance indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001; ns = not significant. Figure 7. Ataxin-2-Targeted Peptide Reduces Astrocyte Activation in TAR4/4 Mice Representative images of GFAP (red) immunostaining in the cortex (A) and lumbar spinal cord (B) of P21 nTg and TAR4/4 (TDP-43 Tg/Tg) mice. Panels show comparisons between nTg saline, TAR4/4 saline, TAR4/4 mice treated with C20-modified peptide, and TAR4/4 mice treated with C16-modified peptide. Merged images of GFAP (red) and DAPI (blue) demonstrate the localization and density of astrocytes in the cortex and lumbar spinal cord in each group. Grey scale images of the selected area show better visualization of GFAP expression. Right panels show quantification of GFAP+ area percentage for each group. Unpaired t test. Data are presented as mean ± SEM, with statistical significance indicated as follows: ****p < 0.0001, ####p < 0.0001; ns = not significant. Supplemental Figure1. Western Blot Analysis of Dose-Response and Time-Dependent Effects of Unmodified Ataxin-2-Targeted Degron Peptides in C57BL6/J Mouse Brain. (A) Dose-response analysis of Ataxin-2 protein levels in brain tissue 4 hours after treatment with increasing doses of the unmodified degron peptide. (B) Time-course analysis of Ataxin-2 degradation in brain tissue following a single 60 mg/kg dose of the unmodified degron peptide. Data are presented as mean ± SEM, n=3 per group, with statistical significance indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Information & Authors Information Version history V1 Version 1 18 June 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Jingyan Zhu The University of British Columbia View all articles by this author Lixia Wang The University of British Columbia View all articles by this author Wendy Wen The University of British Columbia View all articles by this author Ljubomir Kojic The University of British Columbia View all articles by this author Max Cynader [email protected] The University of British Columbia View all articles by this author Metrics & Citations Metrics Article Usage 372 views 272 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jingyan Zhu, Lixia Wang, Wendy Wen, et al. Degron peptide targeting Ataxin-2 mitigates neurodegeneration and neuroinflammation Progression in a TDP-43 Mouse Model of ALS. Authorea . 18 June 2025. DOI: https://doi.org/10.22541/au.175023624.41125652/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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