Synaptic dysfunction induced by glycine‐alanine dipeptides in C9orf72‐ ALS / FTD is rescued by SV 2 replenishment
2020; Springer Nature; Volume: 12; Issue: 5 Linguagem: Inglês
10.15252/emmm.201910722
ISSN1757-4684
AutoresBrigid K. Jensen, Martin H Schuldi, Kevin McAvoy, K. Russell, Ashley Boehringer, Bridget M Curran, Karthik Krishnamurthy, Xinmei Wen, Thomas Westergard, Le Ma, Aaron R. Haeusler, Dieter Edbauer, Piera Pasinelli, Davide Trotti,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoArticle29 April 2020Open Access Source DataTransparent process Synaptic dysfunction induced by glycine-alanine dipeptides in C9orf72-ALS/FTD is rescued by SV2 replenishment Brigid K Jensen Brigid K Jensen orcid.org/0000-0003-1624-6307 Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Martin H Schuldi Martin H Schuldi German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Kevin McAvoy Kevin McAvoy Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Katelyn A Russell Katelyn A Russell Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Ashley Boehringer Ashley Boehringer Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Bridget M Curran Bridget M Curran Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Karthik Krishnamurthy Karthik Krishnamurthy Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Xinmei Wen Xinmei Wen Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Thomas Westergard Thomas Westergard Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Le Ma Le Ma Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Aaron R Haeusler Aaron R Haeusler orcid.org/0000-0001-5566-3707 Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Dieter Edbauer Dieter Edbauer orcid.org/0000-0002-7186-4653 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Piera Pasinelli Piera Pasinelli Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Davide Trotti Corresponding Author Davide Trotti [email protected] orcid.org/0000-0001-6338-6404 Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Brigid K Jensen Brigid K Jensen orcid.org/0000-0003-1624-6307 Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Martin H Schuldi Martin H Schuldi German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Kevin McAvoy Kevin McAvoy Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Katelyn A Russell Katelyn A Russell Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Ashley Boehringer Ashley Boehringer Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Bridget M Curran Bridget M Curran Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Karthik Krishnamurthy Karthik Krishnamurthy Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Xinmei Wen Xinmei Wen Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Thomas Westergard Thomas Westergard Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Le Ma Le Ma Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Aaron R Haeusler Aaron R Haeusler orcid.org/0000-0001-5566-3707 Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Dieter Edbauer Dieter Edbauer orcid.org/0000-0002-7186-4653 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Piera Pasinelli Piera Pasinelli Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Davide Trotti Corresponding Author Davide Trotti [email protected] orcid.org/0000-0001-6338-6404 Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Author Information Brigid K Jensen1, Martin H Schuldi2, Kevin McAvoy1, Katelyn A Russell1, Ashley Boehringer1, Bridget M Curran3, Karthik Krishnamurthy1, Xinmei Wen1, Thomas Westergard1, Le Ma3, Aaron R Haeusler1, Dieter Edbauer2, Piera Pasinelli1 and Davide Trotti *,1 1Jefferson Weinberg ALS Center, Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA 2German Center for Neurodegenerative Diseases (DZNE), Munich, Germany 3Department of Neuroscience, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA *Corresponding author. Tel: +1 215 955 8416; E-mail: [email protected] EMBO Mol Med (2020)12:e10722https://doi.org/10.15252/emmm.201910722 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic hexanucleotide repeat expansion in the C9orf72 gene. In disease, RNA transcripts containing this expanded region undergo repeat-associated non-AUG translation to produce dipeptide repeat proteins (DPRs), which are detected in brain and spinal cord of patients and are neurotoxic both in vitro and in vivo paradigms. We reveal here a novel pathogenic mechanism for the most abundantly detected DPR in ALS/FTD autopsy tissues, poly-glycine-alanine (GA). Previously, we showed motor dysfunction in a GA mouse model without loss of motor neurons. Here, we demonstrate that mobile GA aggregates are present within neurites, evoke a reduction in synaptic vesicle-associated protein 2 (SV2), and alter Ca2+ influx and synaptic vesicle release. These phenotypes could be corrected by restoring SV2 levels. In GA mice, loss of SV2 was observed without reduction of motor neuron number. Notably, reduction in SV2 was seen in cortical and motor neurons derived from patient induced pluripotent stem cell lines, suggesting synaptic alterations also occur in patients. Synopsis Poly-glycine-alanine (GA) aggregates present in neurons cause a reduction in synaptic release and loss of the synaptic vesicle protein SV2 prior to neuronal death. Restoring SV2 levels in these neurons reinstates synaptic release functions and rescues neuronal toxicity. Mobile GA aggregates in neurites of cortical and motor neurons result in increased neuronal Ca2+ influx, inhibition of synaptic vesicle release, and reduction of synaptic vesicle associated protein 2a (SV2). Analysis of transgenic GA149 mice corroborate SV2 reduction in spinal cord and at neuromuscular junctions. Loss of SV2 is seen in C9orf72 patient-derived iPS neurons. Targeted restoration of SV2 levels in GA aggregate-containing neurons normalizes the aberrant Ca2+ influx, impaired synaptic vesicle release, and ultimately rescues neuronal cell death. The paper explained Problem A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of familial and apparently sporadic cases of ALS. While evidence has shown that loss-of-function haploinsufficiency of C9orf72 protein levels may lead to immunological problems, gain-of-function toxic effects caused by accumulation of RNA foci and aggregates of dipeptide repeat proteins (DPRs) both resulting from aberrantly expanded RNA transcripts are the likely pathogenic culprits behind the death of motor neurons. As many C9orf72 patients fall into the sporadic ALS cohort, they possess a high DPR burden by the time of diagnosis. The most abundant of these DPRs is the GA dipeptide, which invokes very gradual cellular toxicity. Results Here, we show that mobile GA aggregates are present in the cytoplasm and axons of cortical and motor neurons. In functional studies, we found that expression of GA resulted in increased Ca2+ influx but reduced synaptic unloading in response to a stimulus for neuronal firing. Examination of synaptic proteins revealed that synaptic vesicle-associated protein 2 (SV2) was specifically reduced. While neurons containing GA ultimately succumbed to toxicity, synaptic protein and transmission deficits occurred several days prior to this demise. Importantly, if we restore SV2 levels in GA-expressing cells, we are able to successfully recover synaptic function and prevent neuronal death. We validated reduction of SV2 in 20-month-old GA transgenic mice, without loss of motor neurons. Finally, we confirmed specific loss of SV2 in C9orf72-ALS patient-derived induced cortical and motor neurons. Impact Understanding and targeting the cellular consequences evoked by the abundant and gradually toxic DPR, GA may be a promising therapeutic option for C9orf72-ALS patients. In isolating molecular mechanisms of synaptic deficits induced by GA, we provide the groundwork for future identification of intervention therapies specifically designed to maintain the synapses required for motor neuron-muscle connectivity and therefore prolong muscle strength in C9orf72-ALS patients. Introduction An intronic hexanucleotide repeat expansion (GGGGCC)n in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (Couthouis et al, 2011; DeJesus-Hernandez et al, 2011; Renton et al, 2011). Evidence has been produced in support of three pathogenic mechanisms linked to this disease-causing expansion: (i) a loss-of-function mechanism due to decreased levels of C9orf72 mRNA expression (Ciura et al, 2013; Therrien et al, 2013; Shi et al, 2018); (ii) RNA transcripts generated from the expanded region gain toxic functions either by forming RNA–DNA hybrids that inhibit transcription and/or by sequestering RNA-binding proteins in nuclear foci (Gendron et al, 2013; Gitler & Tsuiji, 2016); (iii) through repeat-associated non-AUG translation, expanded RNA transcripts encode six dipeptide repeat (DPR) proteins (sense: poly-glycine-alanine or GA, poly-glycine-arginine or GR, poly-glycine-proline or GP; antisense: poly-proline-arginine or PR, poly-proline-alanine or PA, and poly-proline-glycine or PG), which display different toxic profiles in different organismal and cell culture systems (Ash et al, 2013; Mori et al, 2013; Zu et al, 2013; Mizielinska et al, 2014; Haeusler et al, 2016; Wen et al, 2017). Each of these mechanisms invokes widespread cellular responses and, in some cases, culminates in neuronal toxicity and cognitive and motor dysfunctions resembling the human disease phenotype (Kwon et al, 2014; Mizielinska et al, 2014; Wen et al, 2014, 2017; Tao et al, 2015). Evidence is mounting for dysfunctional neuromuscular junction (NMJ) transmission as an early event in C9orf72-ALS (Sareen et al, 2013; Devlin et al, 2015). Notably, two independent studies in Drosophila melanogaster larvae have investigated structural attributes of the NMJ in animals expressing GGGGCC repeats. In these studies, both groups reported drastic reductions in the number of NMJ active zones in larvae expressing 30 or more GGGGCC repeats (Freibaum et al, 2015; Zhang et al, 2015). Detailed analysis also revealed a significant decrease in synaptic bouton number (Freibaum et al, 2015), as well as reduced synaptic quantal content leading to attenuated evoked potentials (Zhang et al, 2015). However, it is not clear whether these effects are due to toxic gain of function of expanded RNA transcripts, DPRs, or a combination of both acting synergistically. Based on numerous recent reports investigating these potential pathogenic mechanisms both in vitro and in animal models, a substantial focus has now been placed on better investigating the effects of DPRs and their potential pathogenic consequences (Gitler & Tsuiji, 2016; Freibaum & Taylor, 2017; Wen et al, 2017). As such, herein we have chosen to limit our mechanistic analysis regarding C9orf72 repeat expansion effects on synaptic function to the contribution of DPR-mediated pathological changes, in the absence of potential confounds due to the concomitant presence of GGGGCC repeat-containing RNA transcripts. Abundant evidence for in vivo and in vitro effects of DPRs has amassed, pointing to pathogenic roles for these dipeptides (Kwon et al, 2014; Mizielinska et al, 2014; Wen et al, 2014; Tao et al, 2015; Boeynaems et al, 2016; Schludi et al, 2017). In fly models as well as primary neurons, arginine-containing DPRs (GR and PR) evoke the most robust toxicity (Mizielinska et al, 2014; Wen et al, 2014; Boeynaems et al, 2016). GR and PR both form aggregates in the nucleus and result in a host of cellular abnormalities including nuclear transport defects, protein mislocalization, altered RNA processing, and nucleolar stress (Kwon et al, 2014; Tao et al, 2015; Kim & Taylor, 2017). While not as overtly toxic, GA also contributes to neuronal dysfunction through ER stress, proteasome impairment, and sequestration of Unc119, HR23, and nucleocytoplasmic transport proteins (May et al, 2014; Zhang et al, 2016). In contrast, marginal or no toxic effects have been attributed to PA and GP/PG (Zu et al, 2013; Wen et al, 2014). While DPR inclusions have been detected throughout the CNS of C9orf72-ALS/FTD patients, the most widespread and abundantly observed are GA+ inclusions (Ash et al, 2013; Gendron et al, 2013; Mori et al, 2013; Zu et al, 2013). In contrast to the robust toxicity resulting from expression of the arginine-rich DPRs, cellular impairments mediated by GA appear to be more subtle. Two mouse models have independently demonstrated motor deficits stemming from neuronal GA expression by 6 months of age. When confined to the cortex, GA50 aggregates lead to neuronal loss, brain atrophy, and mild motor and cognitive behavioral deficits (Liu et al, 2016), whereas when expressed in spinal cord and brainstem, aggregates positive for GA149 trigger significant motor deficits in the absence of overt neuronal loss (Schludi et al, 2017). From this study, our findings suggest that in motor neurons in vivo, GA-mediated synaptic deficits may precede cell death (Schludi et al, 2017). This possibility highlights the necessity for a deeper investigation of subcellular repercussions in neurons coping with expression and aggregation of GA dipeptides, in order to identify mechanisms that may be targeted in a therapeutic window before cell death. We and others have previously demonstrated that GA expression in primary neurons results in reduced neurite outgrowth and cellular toxicity through proteasome impairment and ER stress (May et al, 2014; Zhang et al, 2014). However, the consequences of GA inclusions on neuronal signaling have not yet been examined. In the present study, we have demonstrated that GA aggregates are mobile within axonal and dendritic neuronal compartments. These GA aggregate expressing neurons display disrupted Ca2+ influx, selective down-regulation of the synaptic vesicle-associated protein 2 (SV2), ablated synaptic release, and increased risk of death. Through immunoblot analysis of spinal cords and synaptic staining at neuromuscular junctions from GA149 transgenic mice, which displayed abnormal gait and progressive balance impairment but no appreciable neuronal loss, we have confirmed the selective loss of SV2 expression. Furthermore, we have extended our findings to patient-derived cells, where the full complement of C9orf72 repeat expansion pathogenic mechanisms are potentially at play. In induced pluripotent stem cells differentiated into cortical or motor neurons, neurons derived from C9orf72 repeat expansion carriers again displayed specific reduction of SV2 levels compared with control cells. These in vivo and patient-derived validations of our findings suggest that this molecular phenotype could underlie or contribute to the prodromal progression of disease symptoms. Most crucially, we have been able to restore synaptic function and rescue cellular toxicity through targeted upregulation of SV2 in GA-expressing primary neurons. These findings suggest that GA-mediated alterations of SV2 levels and localization are reversible processes, from which neurons can convalesce, even under the continued presence of GA aggregates. Results GA inclusions are found in axons and dendrites of neurons Immature primary cortical neurons (DIV4) expressing GA149-myc displayed reduced dendritic complexity and sign of apoptosis (May et al, 2014). Nevertheless, in an extensive GA repeat length response curve in mature primary cortical (transfected at 7 DIV) and motor neurons (transfected at 5 DIV), we did not observe significant toxicity at any GA length up to 400 repeats, at 7 or 3 days post-transfection, respectively (Wen et al, 2014). We have also noted in the animal model of GA149-CFP expression that these mice developed behavioral and motor abnormalities, such as abnormal gait and progressive balance impairment in the absence of neuronal loss (Schludi et al, 2017), suggesting dysfunctional neuronal activity. We and others have established that when expressed in a variety of cell lines and in primary rodent neurons, GA forms dense cytosolic aggregates (May et al, 2014; Wen et al, 2014; Zhang et al, 2014). Indeed even when transmitted from cell to cell, GA maintains the tendency for cytosolic accumulation and aggregation (Chang et al, 2016; Westergard et al, 2016). As post-mitotic neurons have distinctive morphology and complex axonal and dendritic arborization architectures, in our prior work we also examined GA-expressing neurons at high spatial resolution (Wen et al, 2014). Mature cortical and motor neurons expressing GA50 (eGFP-tagged) were counterstained with neuronal marker SMI-32. Intriguingly, in addition to cytoplasmic aggregates in the cell soma, eGFP-GA inclusions were also present within neurites (Wen et al, 2014). This localization pattern was unique to the GA dipeptide as we did not observe any of the other dipeptides within neuritic processes (Wen et al, 2014). To begin this present work, in order to determine whether poly-GA aggregates are found in neurites over time, we performed a longitudinal assessment of aggregates, in which GA50 was expressed in mature cortical neurons, with fixation and staining on subpopulations of cells at specific time points. Cells were counterstained with SMI-32. At all time points examined (2, 4, or 8 days of expression), distinctive eGFP+ GA aggregates could be found in neuritic regions (Fig 1). We confirmed that even following 8 days of expression, GFP alone without associated GA peptides does not induce GFP aggregation. Diffuse, cell-filling GFP expression can still be seen in control cells at 8 days of eGFP expression (Fig EV1A). Quantification of the percentage of GA aggregate containing cells displaying neuritic aggregates revealed that at 2 days of expression 66.93% ± 5.57% of cortical GA-expressing neurons have aggregates in their neurites. Comparably, an identical transfection of mature motor neurons revealed that 55.62% ± 6.70% of GA-expressing motor neurons contain neuritic aggregates at 2 days of expression. Next, we wanted to determine the stability of GA aggregates over time. Mature cortical neurons (DIV10) were co-transfected with eGFP, or eGFP-GA50 and a synapsin promoter-driven Td-tomato cell-filling reporter. After 24 h, transfected neurons were identified by co-positivity of eGFP and td-Tomato. The same neurons were then visualized every 24 h for 8 days. We observed that GA inclusions could be cleared over time by the neurons in which they have formed (Fig EV1B). Figure 1. GA50 aggregates are detected in neurites of cortical neurons over timePrimary rat cortical neurons transfected with eGFP-GA50 were examined to determine at which time points preceding cell-death aggregates are found in neurites. Two days post-transfection, aggregates formed by eGFP-GA50 (green) are detectable in neurites (SMI-32 staining in red). These aggregates remain localized to neurites at 96 h (4 days) and 288 h (8 days). Colocalization is indicated by yellow overlay of colors (right panels). Inset below each image shows enlargement of neurite regions containing aggregates, representative fields from 60× magnification z-stack confocal images, scale bar indicates 20 μm. Inset scale bars indicate 5 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. GA aggregates are dynamic A. eGFP was expressed in mature cortical neurons for 8 days and then immunostained for neurofilament (SMI-32, red) and eGFP (green). This representative z-stack confocal images demonstrate cellular viability and lack of GFP aggregation when expressed in the absence of GAn dipeptides even at this extended time. 60× magnification, scale bar indicates 20 μm. B. Primary rat neurons were co-transfected with Td-tomato and eGFP-GA50 plasmid. The same neurons were imaged at 24-h intervals. Representative fields of td-Tomato (top) and eGFP-GA50 (bottom) co-positive cortical neurons at Days 1, 2, 4, 6, and 8 post-transfection follow individual cells over time. Highlighted with green arrows are GA aggregates that dissipate over the course of our imaging period, while the cells containing them remain viable. 20× magnification, scale bar indicates 40 μm. Download figure Download PowerPoint GA aggregates are mobile within neurites The presence of dynamic GA aggregates within neurons suggested additional possible deleterious consequences specific to neuronal cell lineages, including altered cellular trafficking of proteins and organelles to synaptic terminals, as well as dysfunctional synaptic transmission. To probe into these questions, we employed a variety of live-cell imaging functional assays and confocal imaging assessment of synaptic-associated proteins involved in neurotransmission. Using high-resolution live-cell imaging to monitor trafficking of different cargoes within neurites, we first assessed whether the GA aggregates themselves were mobile or stationary, and whether the length of the GA repeat would be a determinant for such mobility. GA at different lengths was transfected into mature cortical or motor neurons at DIV7 and DIV5, respectively. The length range of eGFP-GAn utilized for our current experiments spanned from 25 to 400 repeats as previously described (Wen et al, 2014). The sequences for these constructs were designed following a randomized codon strategy to generate the specified poly-dipeptide sequence of the designated repeat length, but to avoid GGGGCC repeat expansions in the corresponding RNA transcripts (Wen et al, 2014). Brightfield image overlay with the eGFP channel clearly shows distinct GA aggregates within neuritic regions of both cell populations (Fig 2A and B). Following 48 h of expression, high-resolution 60× images were taken at a rapid frame rate, allowing the position of individual eGFP+ GA particles to be tracked over time (NIH ImageJ software). In cortical neurons, aggregates of short GA repeat lengths (25–50) displayed higher mobility along neurites, while aggregates from longer GA repeats (100–400) were more stationary (**P < 0.01; Fig 2C). Similarly, a repeat length-dependent effect on GA particle velocity was observed in primary motor neurons (****P < 0.0001; Fig 2D). We next evaluated whether the presence of neuritic GA aggregates constituted an impediment to the normal trafficking of organelle cargoes along microtubules. To do so, we transfected cultures with eGFP-GAn repeats at increasing lengths and subsequently labeled mitochondria with the cell-permeable dye MitoTracker Deep Red, lysosomes with the cell-permeable dye Lysotracker Deep Red, or total RNA with SYTO RNASelect green. eGFP+ neurons were identified, and mitochondria visualized using the 637-laser channel suitable for the excitation spectrum of the MitoTracker dye. Representative images from cortical neurons demonstrate neuritic eGFP-GA aggregates and distinct mitochondrial labeling with the MitoTracker dye (Fig EV2A). In a similar fashion, eGFP+ neurons were identified, and lysosomes visualized using the 637-laser channel suitable for the excitation spectrum of the Lysotracker dye. Representative images from cortical neurons demonstrate neuritic eGFP-GA aggregates and distinct lysosomal labeling (Fig EV2B). In contrast to the GA aggregates themselves, mitochondrial mobility was unaffected by the presence of GA inclusions within neurites. Mitochondrial mobility was compared across the GA length curve as well as with a GFP-only expressing control, showing no changes at 48 h in cortical neurons (Fig EV2C) or motor neurons (Fig EV2D). Performing the same analysis of lysosomal mobility revealed that only GA400 containing cortical neurons displayed a significant reduction in mobility (*P < 0.01; Fig EV2E). We also assessed the mobility of total cellular RNA through the use of SYTO RNASelect dye labeling in mCherry control or mCherry-GA50 expressing cortical neurons (Fig EV2F). Upon analysis, we again discovered no deficits in overall RNA mobility in GA-expressing cells. We have therefore established that GA aggregates move along cortical and motor neuron processes, without significantly impacting the kinetics of transport of cargoes vital for cell survival. Figure 2. Aggregates from short GA25-50 repeat lengths are mobile, while those from longer repeats are more stationaryPrimary rat cortical and motor neurons underwent live-cell imaging after 48 h of eGFP-GAn expression to determine aggregate mobility within neurites. A. Example of a cortical neuron-expressing eGFP-GA100 (green) with brightfield overlay, 60× magnification, scale bar indicates 10 μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5 μm. B. Example of a motor neuron expressing eGFP-GA25 (green) with brightfield overlay, 60× magnification, scale bar indicates 10 μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5 μm. C. Quantification of eGFP-GAn aggregate velocity within cortical neuron neurites. D. Quantification of eGFP-GAn aggregate velocity within motor neuron neurites. Data information: Data presented as mean ± SEM. One-way ANOVA, post hoc Dunnett's multiple comparison test, **P < 0.01, ****P < 0.0001. Exact P-values can be found in Appendix Table S1. Velocity measurements from 10 aggregates were assessed from each of three biological replicates for each GA repeat length in each neuronal population. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Cellular trafficking is unaffected by the presence of neuritic GA aggregatesCortical and motor neurons underwent live-cell imaging after 48 h of eGFP-GAn peptide expression to determine mobility of cargoes within neurites. MitoTracker Deep Red FM dye (50 nM)-labeled mitochondria, LysoTracker Deep Red (50 nM)-labeled lysosomes, SYTO RNASelect-labeled total cellular mRNA. A. Example of a cortical neuron expressing eGFP-GA50 (green) with labeled mitochondria (magenta). Right
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