2023 Jefferson Synaptic Biology Symposium Abstracts
1. Divergent activity-dependent transcriptome and regulation of the C9orf72 gene in ALS-FTD neurons
Layla Ghaffari1, Emily Welebob1, Davide Trotti1, Aaron Haeusler1
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders with significant clinical overlap and a common genetic cause: a hexanucleotide repeat expansion mutation in the C9orf72 gene. The functions of C9orf72 in the nervous system are becoming increasingly apparent. Still, it is unknown whether C9orf72 can be regulated in a neural-specific manner or how the repeat expansion mutation leads to global alterations of the neuronal transcriptome. Neuronal activity is a crucial modifier of biological processes in health and neurodegenerative disease contexts. Our studies have shed light on the activity-dependent regulation of C9orf72 gene expression in rodent and human neurons, with evidence showing that membrane depolarization can modulate the expression of C9orf72 transcript variants. Furthermore, we used RNA sequencing to investigate the activity-dependent transcriptome of neurons derived from patients with C9orf72-linked ALS/FTD. We found that patient-derived neurons display a divergent transcriptional response to depolarization compared to control neurons. Specifically, we observed the dysregulation of genes involved in synaptic function, ion channel activity, and the extracellular matrix. Understanding the mechanisms underlying the dysregulation of the C9orf72 gene and activity-dependent gene expression in C9orf72-linked ALS-FTD neurons could provide novel therapeutic targets for the treatment of these devastating diseases.
2. Proteomic profiling of autophagosomes in healthy and Parkinson’s disease model brain uncovers novel regulation of mitochondrial DNA in neurons
Juliet Goldsmith, Alban Ordureau, J Wade Harper, Erika LF Holzbaur
Neurons depend on autophagy to maintain cellular homeostasis, while defective autophagy and impaired mitochondrial maintenance are pathological hallmarks of neurodegenerative diseases. In order to better understand the role of autophagy in maintaining neuron health, we established a reliable method for enriching autophagic vesicles (AVs) from mouse brain and used proteomics to identify the major cargos engulfed within. We identified pre- and post-synaptic proteins as basal cargo, suggesting homeostatic autophagy mainly occurs at or near synapses. We additionally observed small, undamaged mitochondria within the AVs that are enriched for mitochondrial DNA and its associated protein TFAM. Thus we describe novel mitochondrial regulation by autophagy that is distinct from the clearance of damaged mitochondria by mitophagy. We further compared the cargos in brain-derived AVs from two different genetic models of Parkinson’s disease (PD), and find distinct upregulation of pathways to complement impaired autophagy. In the PINK1-/- model in which mitophagy is disrupted, there are increased levels of alternative mitophagy receptors, as well as higher levels of key proteins for autophagosome biogenesis, and maintained mitochondria engulfment. In contrast, the LRRK2G2019S model in which cargo degradation is impaired has increased secretion of EVs and TFAM in conditioned media from LRRK2G2019S neurons and in serum from LRRK2G2019S mice. Whether increased secretion of autophagy cargo mitigates damaged protein and organelle accumulation in the neuron, or results in increased inflammation and pathological aggregate spreading is an on-going area of research. Our combined evidence suggests that the autophagic regulation of mitochondrial DNA is protected in neurons.
3. Investigating How Map7 Coordinates Motor Recruitment To Regulate Transport Selectivity At Branch Junctions
Elizabeth R. Moese, Stephen R. Tymanskyj, Le Ma
Proper development and function of neurons requires tightly regulated microtubule (MT) -based transport of various cargo from cell bodies to synaptic terminals. Improper transport is implicated in various neurological diseases. Neurons have elaborate branches creating complex connections. Transport at branch junctions requires tight regulation of cargos, as suggested by recent discovery of selective transport in cultured DRG neurons. Furthermore, this selectivity is differentially regulated for various cargos and mediated by kinesin-3 motors. However, it is unknown what local mechanisms at branch junctions are mediating transport selectivity. Interestingly, MAP7 is a MT associated protein enriched at branch junctions in embryonic DRG neurons. In vitro, MAP7 recruits kinesin-1 to MTs but inhibits other kinesin-3 from MT binding. Transport of cargos such as mitochondria in the presence of MAP7 displayed increased speed switching suggesting motors can switch MT tracks. We hypothesize that MAP7 coordinates motor recruitment and transport selectivity at branch junctions. To test this, we examined MAP7 knockout neurons and preliminary data showed a decrease in mitochondria speed switching consistent with the idea that locally MAP7 is coordinating motor recruitment. MAP7 can be phosphorylated which may affect transport selectivity at branch junctions. Mutating SP/TP sites in the P-domain, affects MT binding and localization. These data suggest phosphorylated MAP7 is more readily removed from MTs and alters localization which might result in changes in transport at branch junctions, specifically by increasing kinesin-3 transport through branches. Finally, with expansion microscopy we will examine MAP7 localization at branch junctions at a higher resolution.
4. Synaptic dysfunction in MICU1KO neurons is associated with uncontrolled mitochondrial calcium buffering. – POSTER AWARD
Raghavendra Singh, Adam Bartok and György Hajnóczky
Mitochondrial clearance of intracellular calcium and ATP production during neuronal stimulation play a crucial role in neuronal health, and modulating Ca2+ dependent synaptic function. Ca2+ enters the mitochondrial matrix via the mitochondrial Ca2+ uniporter complex, the pore-forming unit of which is MCU. Ca2+ sensitivity to MCU is conferred by MICU1, a mitochondrial intermembrane space protein that helps to keep MCU closed at low [Ca2+] and supports MCU’s cooperative activation as [Ca2+] rises. Human loss of function mutation of MICU1 has been linked to learning difficulties, skeletal muscle weakness, motoric impairment and fatigue. However, the neuronal basis of the pathomechanism is unknown. In the present study, using neuron-specific MICU1 KO mice and culturing full body MICU1 KO E15.5 primary cortical neurons, we demonstrated the loss of the gatekeeping and cooperative activation by Ca2+ in mitochondrial Ca2+ uptake, and identified mitochondrial Ca2+ overload as an inducer of cell death. We further asked, if MICU1 regulation of mitochondrial calcium entry influences the local synaptic intracellular calcium buffering and neurotransmitter-vesicle fusion during synaptic activity. We found WT and KO neurons with similar global cytoplasmic [Ca2+] ([Ca2+]c) responses during electrical stimulation (ES). In KO neurons, mitochondrial matrix [Ca2+] ([Ca2+]m) rise was ensued even at 50nM increase in [Ca2+]c upon low pulse (10-30) field stimulation (20V, 10Hz), and it was tightly coupled to the [Ca2+]c increase. In WT neurons, high pulse (150) field stimulation (40V, 20Hz) was required for a [Ca2+]m rise, and it lagged behind the [Ca2+]c rise. Furthermore, at the sub-optimal field stimulation the presynaptic local [Ca2+]c rise was attenuated by the presence of mitochondria in KO neuron but not in WT. Also, the cytoplasmic ATP:ADP ratio showed a tendency for lower resting ATP/ADP and a larger decrease during ES in KO neurons. Moreover, synaptic (SV) fusion was suppressed in KO neurons at all the ES parameter used, particularly, the secretory response evoked by repetitive suboptimal ES seemed to be greatly inhibited in KO neurons. Notably, the ES-evoked SV fusion at the boutons lacking mitochondria was not different among WT and KO, while mitochondrial presence suppressed SV fusion immensely in KO. In addition, blocking reacidification of alkaline trapped vesicles by bafilomycin-A1 led to a larger maximal ES-evoked secretory response in WT than KO, suggesting that exocytosis per se is affected in MICU1 KO neurons. Thus, our results highlight the specific contribution of MICU1-gated mitochondria in the regulation of Ca2+-dependent exocytosis of neurotransmitter vesicles by modulating local intracellular Ca2+ levels at the neuronal processes. Altogether, our study proposes a neuronal pathomechanism that might underlie behavioral dysfunction in MICU1 loss of function patients.
5. WHITDRAWN
6. Regulation of Transport Selectivity at DRG Neuron Branch Junctions
Siddharth Karthikeyan, Stephen Tymanskyj, Le Ma
The complex, branching geometry of neurons poses a challenge for the efficient trafficking of intracellular components throughout the cell. While many of the molecular mediators of transport (i.e. cytoskeletal tracks, motor proteins, MAPs, etc.) have been identified, further investigation is required to understand the variable regulation of these proteins in the different subcellular compartments of the cell. In particular, little is known about transport regulation at branch junctions, which are physiologically significant sites that allow neurons to extend processes to multiple targets and participate in complex networks. Prior work from our lab has shown that selectivity of anterograde lysosomal transport at these sites (i.e. whether the organelle is moved to one branch or another competing branch at the junction) correlates with branch length and growth cone motility. Here, we present optogenetic data that suggests modulation of growth cone activity through (1) induction of canonical neurotrophic/neurotropic signaling and (2) steady depolarization can also affect the selectivity of lysosomal transport at branch junctions.
7. Intracerebral transplantation of autologous bone marrow stem cells produces functional recovery in rats with long-term stable strokes.
Max I Myers, Kevin J Hines, Gabrielle Spagnuolo, Andrew Gray, Yolanda Gomez-Galvez, Jingli Cai, Robert Rosenwasser, Lorraine Iacovitti
While treatment options exist for the acute phase of stroke, there are limited options for patients with stable infarcts and long-term disability. Allogenic mesenchymal stem cells (MSCs) have shown promise for the treatment of stroke when delivered systemically soon after ischemic injury. There is, however, limited data on the use of a) autologous MSCs, b) delivered via subcortical stereotactic transplantation c) in rats with a stable infarct. This study seeks to evaluate the efficacy of intracerebral transplantation of autologous MSCs in a rat middle cerebral artery occlusion (MCAO) model of chronic stroke. Male Sprague-Dawley rats underwent right middle cerebral artery occlusion for 120 minutes to induce stroke. 16 days following stroke, rats underwent tibial bone marrow aspiration. Autologous MSCs were then cultured and expanded in a closed-system sterile growth bioreactor. 1 month following stroke, MSCs were harvested, brain MRI was obtained, and a stereotactic injection robot was used to implant various doses of stem cells via three trajectories in the peri-infarct region. The total number of cells in the three treatment groups consisted of (1x10^6 cells, 2.5x10^6 cells, 5x10^6 cells, n=6 in each group). The control group received an MCA stroke and was saline-injected (n=9). In a second cohort of animals, fluorescent tagged quantum dots (QD) were used to label autologous MSCs, which were tracked in the brain at 1 week, 1 month, and 2 months post-transplantation. Behavior was assessed using the modified neurological severity score (mNSS) (0-16), revealing highly significant neurological improvement at 1 and 2 months following MSC transplantation in all treatment animals, compared to controls. No apparent dose response in efficacy measures was observed since 1X10^6 cells was likely beyond the threshold needed for treatment efficacy. As expected, no difference in terms of ischemic lesion volume or MRI aspect were observed between MSC-treated and control animals, as cells were transplanted 1 month after the acute injury of MCAO. Immunocytochemistry revealed increased astrocyte and microglia reactivity along the peri-infarct region. Surprisingly, quantum dot analysis displayed the continued long-term presence of MSCs in the MCAO brain, possibly increasing their long-term effectiveness. Thus, these studies suggest that intracerebral transplantation of autologous MSCs may be a promising treatment for chronic MCA stroke.
8. Selective axonal transport through branch junctions is directed by growth cone signaling and mediated by KIF1/kinesin-3 motors – SELECTED TALK
Stephen R Tymanskyj, Bridget M Curran, Le Ma
Development of a functioning nervous system requires the establishment of individual neurons communicating with multiple targets, achieved by a single axon generating multiple branches. These branches differ in lengths and targets, and as such have different metabolic and protein needs. Delivery of proteins from the cell body to the correct targets rely on the orchestration of microtubule-based transport by the motor protein kinesin family, but if and how protein or membrane cargos are regulated along the axon is unknown. Here we demonstrate that cargo delivery is in fact regulated along the axon, specifically through branch junctions where they can target specific branches. We show that anterograde transport of LAMP-1 and synaptic vesicles through axonal branch junctions is highly selective, influenced by branch length and more strongly by growth cone motility. We further show that not all cargos are responsive to the same cues with secretory vesicles such as BDNF being largely unresponsive. We developed an optogenetic tool based on mimicking the guidance receptor activity of positive (TrkA) and negative (PlexinA4) cues and focally activated one specific growth cone. We were able to demonstrate that signaling from the growth cone can rapidly direct transport through branch junctions located a distance away from the site of activation. We further demonstrate that such transport selectivity is differentially regulated and mediated by the KIF1/kinesin-3 family motors. We propose that this transport regulation through branch junctions could broadly impact neuronal development, function, and regeneration.
9. WHITDRAWN
10. The structural basis of the mechanism of action of highly specific positive allosteric modulators of Kv3 channels
Qiansheng Liang,1,2 Lianteng Zhi,1,2 Leonardo Cirqueira,3 Gamma Chi,4 Werner Treptow,3 Manuel Covarrubias1,2
Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat severe neurological disorders. However, the development of selective modulators requires an understanding of their mechanism-of-action (MoA). We applied an orthogonal approach to elucidate the MoA of an imidazolidinedione derivative (AUT5), which is a highly specific positive allosteric modulator (PAM) of Kv3.1 and Kv3.2 channels. With an EC50 of 3.2 mM, AUT5 induces cooperative positive modulation by preferentially stabilizing the open state. Also, the unique and highly conserved extracellular turret region of Kv3.1 and Kv3.2 was found to be the essential determinant of AUT5 action. Furthermore, leveraging on the cryo-EM structure of Kv3.1a, atomistic blind docking calculations revealed four equivalent AUT5 binding sites near the turrets and between the voltage-sensing and pore domains of the channel’s tetrameric assembly. Directly validating these results, a new cryo-EM structure of Kv3.1a precisely resolved the binding site of AUT1 (another imidazolidinedione PAM closely related to AUT5) and relevant interactions at the location predicted by the docking calculations. Therefore, the unique Kv3 turret emerges as a novel structural correlate of the selective MoA of a new class of Kv3 channel PAMs with a therapeutic potential.
11. WITHDRAWN
12. Mitochondrial structure in neuronal processes of healthy and degenerating brain
David Weaver, Raghavendra Singh, Prashant Badgujar, Aron Andresi, Gyorgy Csordas, Gyorgy Hajnoczky
Mitochondrial energy production and calcium handling is central to the normal neuronal function, including neurotransmission. Mitochondria are commonly closely positioned to and involved in local control of ATP and calcium at both the pre-synaptic and the post-synaptic sites. However, mitochondrial positioning relative to the synaptic machinery, mitochondrial shape and internal structure in the brain have been difficult to study. To fill this gap, we used Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) that allows 3 nm x-y-z resolution in large volumes, and machine learning approaches to determine mitochondrial ultrastructure in the prefrontal cortex of adult mice. We have studied both normal mice and mice with neuron-specific deletion of MICU1, the gatekeeper of mitochondrial calcium uptake. The MICU1-deficient mice and MICU1 loss of function patients display neurodegeneration dominated by motor function and learning/memory impairments. Our presentation will focus on mitochondrial shapes in both axons and dendrites and on clues to their alterations in MICU1 loss related neurodegeneration. We will also display the inner mitochondrial membrane complexity resolved at 3nm in entire mitochondria of normal and MICU1-deficient axons and dendrites for the first time. We will interpret the new structural information in the context of mitochondrial and synaptic function and dysfunction.
13. Histone variant H2BE promotes chromatin accessibility, synaptic gene expression, and long-term memory
Emily Hyatt, Sean Louzon, Kate Palozola, Qi Qiu, Kyuhyun Choi, Nicholas Prescott, Yael David, Marc Fuccillo, Hao Wu, Erica Korb
14. Phosphorylation-dependent cumulative inactivation of Kv3.4 channels governs use-dependent action potential broadening in DRG neurons – POSTER AWARD
T. Alexander, A. Alicea-Pauneto, S. Tymanskyi, M. Covarrubias
Kv3.4 channels can tune the repolarization rate of the DRG neuron action potential (AP) in a manner that depends on the phosphorylation status of their cytoplasmic N-terminal inactivation domain (NTID) at four non-equivalent serines. However, the mechanism underlying this link is unknown. Based on computational modeling, we hypothesize that use-dependent AP broadening depends on Kv3.4 recovery from inactivation, which, in turn, depends on the phosphorylation status of the NTID. To test this hypothesis, we used an AAV6-based approach in rat DRG neurons to overexpress wild-type Kv3.4 (susceptible to basal phosphorylation) and mutant Kv3.4 channels that were either dephosphorylated (Ala mutations = phospho-null) or fully phosphorylated (Asp mutations = phosphomimic) at four serines and characterized the expressed Kv3.4 currents and APs under repetitive stimulation conditions that induce cumulative inactivation and use-dependent AP broadening. We found that that robust use-dependent AP broadening is associated with profound cumulative inactivation and slow recovery from inactivation of the phosphonull Kv3.4. In contrast, modest AP broadening is associated with less severe cumulative inactivation and faster recovery from inactivation of the wild-type Kv3.4. Strikingly, when overexpressing the phosphomimic Kv3.4, use-dependent AP broadening and cumulative inactivation are nearly inhibited – which is associated with faster recovery from inactivation. These results establish the molecular mechanism responsible for the regulation of use-dependent AP broadening in DRG neurons, which plays a central in the transmission of nociceptive signals at the level of the first synapse in the dorsal horn of the spinal cord.
15. Acute in vivo expression of ALS-related proteins: opportunities for rapid assessment of genetic variants, cell type contributions, and potential therapeutics
Brigid K. Jensen, Jenny Carrey, Matt Davis, Lin Guo, Piera Pasinelli
Amyotrophic Lateral Sclerosis (ALS) is a progressive, adult-onset neurodegenerative disease characterized by loss off upper motor neurons in the cortex and lower motor neurons in the spinal cord. It is a complex, heterogenous disease with a myriad of disease-causative genetic variants. Moreover, while ALS ultimately targets motor neurons, non-neuronal cells such as astrocytes and microglia have also been demonstrated to be important drivers of disease pathology. Thus, to study mechanisms underlying ALS, we utilize in vitro cell culturing methods paired with rodent in vivo modeling approaches through expression of ALS-causative proteins. However, typically, creation of animal models is very time and cost intensive. As a result, for many ALS genetic variants, no ubiquitously expressing in vivo model has been generated. Rarer still are targeted models designed to study the contributions of specific cell populations to disease pathogenesis and progression. An additional, independent frustration in ALS research has been that many potential therapeutic compounds have shown efficacy when tested in vitro, yet fail when applied to animal models or human clinical trials. Therefore, a quick in vivo screening approach to determine target engagement and intended pharmacological effect would streamline design of more comprehensive studies. Therefore, it was the goal of this project to utilize acute spinal cord expression of ALS-related proteins as a method to quickly and accurately assess pathogenicity of genetic variants, cell-type mediated effects, and potential of therapeutic compounds in vivo.
16. MICU2 Loss is Associated with Altered Mitochondrial Calcium Signaling in the Nervous System during Development
Elena Berezhnaya, Benjamín Cartes Saavedra, Raghavendra Singh, Macarena Rodriguez-Valle, Fowzan S. Alkuraya, Gyorgy Hajnoczky
Neurological disorders are often accompanied by mitochondrial dysfunction and calcium overload. Calcium enters mitochondria via mitochondrial calcium uniporter complex (mtCU) consisting of the pore-forming subunit, MCU, scaffold protein, EMRE, and regulatory subunit, MICU1, either homodimerized or heterodimerized with MICU2 or MICU3. MICU1 loss was shown to promote mitochondrial calcium overload and consequent neurodegeneration in mice and patients. MICU2 null mutation in patients leads to neurodevelopmental disorder, but the underlying mechanism is unclear. We used fluorescent live-cell imaging of patient fibroblasts and found that MICU2 loss results in higher resting [Ca2+]m and elevated mitochondrial calcium uptake following similar [Ca2+]c rises evoked by depletion of endoplasmic reticulum and induction of store-operated calcium entry. However, based on MitoCarta 3.0, brain has very little MICU2 with MICU1 and MICU3 being prevalent. To study if MICU2 affects mitochondrial calcium handling in the nervous system, we generated MICU2 -/- mice using Cre/loxP strategy and cultured MICU2 -/- and MICU2loxP primary neurons. We confirmed MICU2 presence in neurons and examined their mitochondrial calcium signaling by fluorescent live-cell imaging. Similar to patient fibroblasts mitochondria in MICU2-deficient neurons start taking up calcium at lower [Ca2+]c during electrical stimulation, however their resting [Ca2+]m is unaltered. We further confirmed MICU2 presence in the mouse brain and showed that its abundance decreases during brain development almost disappearing by day 21. Thus, the MICU2 role in the nervous system seems to be limited to early development and its loss promotes mitochondrial calcium overload that might be a cause of neurodevelopmental impairments in patients.
17. Synaptic development in diverse olfactory neuron classes uses distinct temporal and activity-related programs
Michael A. Aimino, Alison T. DePew, Lucas Restrepo, and Timothy J. Mosca
Developing neurons must follow specific molecular, cellular, and temporal programs to establish synapses capable of comprising a functional circuit. However, the vast diversity in class, morphology, and function of brain neurons raises important questions. Do all classes of neurons use the same, or different, organizational mechanisms to form synapses? Furthermore, do neurons develop on similar timescales and use identical molecular approaches to form the correct number of synapses? To investigate these questions, we used the Drosophila antennal lobe, a model olfactory circuit with remarkable genetic access and synapse-level resolution. We performed a quantitative analysis of synapse formation in multiple classes of olfactory neurons throughout development and adulthood and found that olfactory receptor neurons (ORNs), projection neurons (PNs), and local interneurons (LNs) each have unique time-courses of synaptic development, addition, and refinement, demonstrating that each class follows a distinct developmental program. This raised the possibility that these classes may also have distinct cellular requirements for synapse formation. Therefore, we genetically altered neuronal activity in each class of neuron and found that silencing neuronal activity in ORNs, PNs, and LNs impaired synaptic development but only in ORNs did enhancing neuronal activity influence synapse formation. Intriguingly, ORNs and LNs demonstrated similarly impaired synaptic development with overexpression of a constitutively active version of the master kinase, GSK-3β, suggesting that neuronal activity and GSK-3β function in a common pathway. Ultimately, our results suggest that the requirements for synaptic development are not uniform across all neuronal classes, providing novel insights into the mechanisms of synaptic development.
18. Absence of chordin-like 1 prevents loss of GluA2 and improves motor recovery in a mouse model of stroke.
Bridget R Boyle, Eileen Collyer, Yolanda Gomez-Galvez, Lorraine Iacovitti, Elena Blanco-Suarez.
Chordin-like 1 (Chrdl1) is an astrocyte-secreted protein that regulates synaptic maturation, and limits plasticity. In response to ischemic stroke, Chrdl1 is upregulated during the acute and sub-acute phases in the peri-infarct region, potentially hindering recovery after stroke. It was demonstrated that Chrdl1 is enriched in astrocytes in cortical layers 2/3, with peak expression in the visual cortex at postnatal day 14. Here, we used photothrombotic stroke to target the motor cortex of adult male and female mice. In this study, we demonstrate that elimination of Chrdl1 in a global knock-out mouse prevents ischemia-driven synaptic loss of the GluA2 AMPA receptor subunit, reduces apoptotic cell death and promotes faster motor recovery. This suggests that synapse-regulating astrocyte-secreted proteins such as Chrdl1 have therapeutic potential to aid functional recovery after an ischemic injury.
19. Loss Of Dot1l And H3k79 Methylation Impacts Long-Term Memory And Synaptic Gene Expression In Neurons
Marissa Maroni, Melissa Barton, Samuel Thudium, Katherine Lynch, Rachel Lee, Karim-Jean Armache, Erica Korb
20. Glucose Hypometabolism Prompts RAN Translation and Exacerbates C9orf72-related ALS/FTD Phenotypes
A.T. Nelson1, M.E. Cicardi1, S.S. Markandaiah1, J. Han2, N. Philp2, E. Welebob1, A.R. Haeusler1, P. Pasinelli1, G. Manfredi4, H. Kawamata4 and D. Trotti1
The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, although its potential role in disease pathogenesis is unknown. Here, we identified alterations in glucose metabolic pathways and ATP levels in the brain of asymptomatic C9-BAC mice. We found that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also found that one of the arginine-rich DPRs (PR) can directly contribute to glucose metabolism and metabolic stress. These findings provide a mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and support a feedforward loop model that opens several opportunities for therapeutic intervention.
21. The cell surface receptor LRP4 promotes synaptic growth, active zone organization, and synaptic maturation through a downstream SR-protein kinase mechanism.
Alison T. DePew, Joseph Bruckner, Kate O’Connor-Giles, Timothy J. Mosca
22. Spastin locally amplifies microtubule polymerization to pattern the axon for presynaptic cargo accumulation – SELECTED TALK
Jayne Aiken and Erika L. F. Holzbaur
Neurons rely on long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, and how efficient delivery is maintained, remains largely unknown. Using human neurons derived from induced pluripotent stem cells (iPSCs), we reveal a novel role for the microtubule severing enzyme spastin in locally amplifying microtubule mass to influence presynaptic cargo pausing and retention along the axon. Even in the absence of robust synaptic connections, “protosynaptic” sites inhabited by stable synaptic vesicle precursors (SVPs) are hotspots for dynamic microtubules and SVP pausing in both the anterograde and retrograde directions. Disruption of neuronal Spastin, either by depletion via CRISPR interference (CRISPRi) or by transient overexpression, interrupts the localized enrichment of microtubule amplification and diminishes SVP accumulation at protosynapses. To further interrogate Spastin’s role in presynaptic cargo delivery, we developed a heterologous synapse protocol utilizing microfluidically isolated human axons and neuroligin-expressing HEK cells to rapidly induce robust presynapse formation. This novel method provides unique spatial and temporal control over human presynapse formation, and revealed that axonal spastin enhances presynaptic retention. We propose a model where Spastin acts locally as an amplifier of microtubule polymerization to pattern the axon for synaptogenesis and guide synaptic cargo delivery.
23. Regulation of striatal circuit formation by the nuclear-localized protein Zswim6
N.T. Henderson, K. Choi, D. Tishfield, S.A. Anderson, E. Korb, M.V. Fuccillo
The striatum integrates synaptic inputs from multiple brain regions and is crucial for many motor and cognitive processes. Yet, relatively little is known about the genetic and molecular mechanisms governing striatal circuit development and maintenance. Zswim6 (Zs6) is a striatally expressed gene that is implicated in Schizophrenia and intellectual disability. The function of Zs6 is unknown, but the presence of SWIM and sin3-like domains suggest a role in gene regulation. Here, we find that exogenously expressed Zs6 associates with chromatin in heterologous cells and localizes robustly to the nucleus in heterologous cells and neurons. Consistent with the known role of sin3 domains in transcriptional repression, we found that conditional knockout of Zs6 in striatal GABAergic progenitors increases chromatin accessibility, as assessed by ATAC-seq. Using single nucleus RNA-seq, we show that deletion of Zs6 alters expression of genes related to synaptic transmission, synapse development, and membrane excitability. Conditional deletion of Zs6 in SPNs did not affect cell morphology, but impacted synaptic function. Zs6 deletion in either direct pathway or indirect pathway SPNs caused an increase in paired-pulse ratio, resulting from reduced desensitization of AMPARs. AMPA/NMDA ratios and mEPSC amplitudes were also reduced, further suggesting widespread dysregulation of AMPAR signaling. Conditional deletion of Zs6 in adulthood recapitulated these synaptic phenotypes, indicating that continued expression of Zs6 in adulthood is required for maintaining synaptic function. Future experiments will involve identifying genes directly regulated by Zs6 using ChIP-seq, and examining circuit-level and behavioral consequences of synaptic dysfunction resulting from Zs6 loss-of-function.
24. Sensory Experience Stimulates Neuron-Astrocyte Sonic Hedgehog Signaling – POSTER AWARD
Anh Duc Le, Steve Hill Ph.D., Marissa Fu, Pooja Sakthivel, Joshua Mell Ph.D., A. Denise R. Garcia Ph.D.
Astrocytes, the most abundant subtype of glial cells in the CNS, play a crucial role in regulating the formation and function of neuronal synapses. Growing evidence suggests astrocytes can facilitate experience-dependent synaptic plasticity, however the mechanism by which they accomplish this is poorly understood. The Sonic hedgehog (Shh) signaling pathway is a compelling candidate for mediating this bidirectional communication. In the adult cortex, Shh is expressed by neurons localized to layer V and transduced by a subpopulation of layer IV and V astrocytes. To examine whether Shh signaling can be regulated by neuronal activity, we housed mice in an enriched environment to promote robust somatosensory activity. We found that enriched sensory experience stimulated Shh signaling in the somatosensory, but not motor and visual cortex, suggesting that experience-dependent Shh signaling occurs in a modality-specific manner. Chemo-genetic activation of neurons confirmed neuronal activity alone can stimulate Shh activity. Next, to identify potential activity-dependent genes regulated by Shh signaling, we performed bulk RNA sequencing to identify genes enriched in Shh-transducing astrocytes. We identified SPARC and Hevin – astrocyte-secreted matricellular proteins that regulate synaptic formation, function and plasticity – as selectively enriched in Shh-transducing astrocytes compared to the total cortical astrocyte population. Disruption of Shh signaling in astrocytes reduced Sparc and Hevin, identifying these genes as Shh-dependent. Sensory enrichment increased SPARC and Hevin abundance, identifying these proteins as experience-dependent. In summary, our work revealed a novel activity-dependent feature of neuron-astrocyte Shh signaling, deepening our understanding of neuron-astrocyte bidirectional communication in experience-dependent plasticity.
25. Modulation and Neural Correlates of Postmating Sleep Plasticity in Drosophila Females
José M. Duhart, Joseph R. Buchler, Sho Inami, Kyle J. Kennedy, B. Peter Jenny, Dinis J.S. Afonso, Kyunghee Koh
Sleep is essential, but animals may forgo sleep to engage in other critical behaviors, such as feeding and reproduction. Previous studies have shown that female flies show decreased sleep after mating, but our understanding of the process is limited. Here, we report that postmating nighttime sleep loss is modulated by diet and sleep deprivation, demonstrating a complex interaction among sleep, reproduction, and diet. We also report that female-specific pC1 neurons and sleep-promoting dorsal fan-shaped body (dFB) neurons are required for postmating sleep plasticity. Activating pC1 neurons leads to sleep suppression on standard fly culture media but has little sleep effect on sucrose-only food. Published connectome data suggest indirect, inhibitory connections among pC1 subtypes. Using calcium imaging, we show that activating the pC1e subtype inhibits dFB neurons. We propose that pC1 and dFB neurons integrate the mating status, food context, and sleep drive to modulate postmating sleep plasticity.
26. The role of Extracellular Tyrosine Kinase VLK in synaptic recruitment of NMDAR
Praveen Chander, Srikanth D Kolluru, Halley R Washburn, Theodore J Price, Matthew B Dalva
NMDAR synaptic localization at cortical spine synapse is governed by the EphB family of receptor tyrosine kinases. EphB2 controls NMDAR synaptic localization and function via a direct interaction between the extracellular fibronectin-type III domain on EphB2 and the N-terminal domain of GluN1. This direct interaction is mediated by a seemingly novel mechanism – the phosphorylation of a specific, and highly conserved tyrosine residue in Y504 on EphB2. While protein function modification by intracellular phosphorylation is one of the more widely appreciated mechanisms mediating protein-protein interactions, we propose that extracellular phosphorylation of proteins may play a similar role. A key question to be addressed is whether there are specific kinases that mediate these events. Extracellular phosphorylation is mediated by twelve extracellular kinases, six directed at serine/threonine and six directed at tyrosine. These kinases are linked to a diverse set of diseases including those related to bone development, autism spectrum disorder, and other diseases of brain dysfunction. Because the EphB-NMDAR interaction requires tyrosine phosphorylation, we focused on examining the role of the six tyrosine kinases.Our previous work showed that phosphorylation of Y504 occurs on the cell surface, therefore we tested whether any of the six kinases might be able to induce the EphB-NMDAR interaction when added exogenously to cells. When transfected into HEK293T cells or applied to HEK293T cells or neurons, only exogenous VLK/PKDCC induced EphB-NMDAR. Kinase dead VLK mutant protein failed to induce the interaction. Moreover, knockdown of VLK expression blocked the EphB-NMDAR interaction. These data indicate that VLK is necessary and sufficient for the EphB-NMDAR interaction. We next asked where VLK was localized in the brain. Synaptosome fractionation revealed that VLK is enriched in the synaptic vesicle fraction. In vitro, VLK co-transports in axons with Synaptophysin 1 (SYP1) and co-localizes with SYP1 and PSD-95 in axons, suggesting that VLK is found at presynaptic sites. These data suggest that VLK might be secreted from neurons using a mechanism similar to synaptic release. To begin to test this we asked if ephrin-B2 activation of EphB2 might induce VLK release from neurons. Ephrin-B induced VLK secretion from cortical neurons. Next we asked whether inhibition of SNARE dependent release might inhibit VLK secretion. Remarkably, pre-treatment of neurons with Botulinum toxin A blocked VLK secretion. These data suggest that VLK release is regulated by EphB signaling and VLK is necessary and sufficient to mediate EphB-NMDAR interaction.
27. EphBs and Their Role in Spinal Cord Injury-Induced Neuropathic Pain
David A. Jaffe, Nicolette M. Heinsinger, Kolluru D. Srikanth, Rachel E. Cain, R. Vivian Allahyari, Lan Cheng, Jaime L. Watson, Aditi Falnikar, Wei Zhou, Eric V. Brown, Brittany A. Charsar, Michael E. Greenberg, Matthew B. Dalva, Angelo C. Lepore
A major portion of individuals with spinal cord injury (SCI) suffer from debilitating chronic neuropathic pain (NP). Central sensitization, or the hyperexcitability of CNS pain circuitry, is a major substrate for SCI-induced NP and can include alterations to NMDA receptor (NMDAR) signaling in the spinal cord dorsal horn (DH). In a mouse model of cervical contusion-type SCI that produces both evoked and spontaneous NP-related behavior, we found increased EphB2 gene and protein expression in DH, as well as enhanced colocalization of EphB2 and GluN1 (the obligate NMDAR subunit) at vGlut-positive sites in superficial DH neurons, suggesting an enhanced EphB2-NMDAR interaction at putative excitatory synapses. Furthermore, in situ hybridization analysis revealed upregulated EphB2 expression in superficial DH after SCI, including specifically in the NK1R-expressing population of projection neurons marked by the tacr1 gene. Targeted inducible inhibition of the intracellular tyrosine kinase activity of EphB1, EphB2 and EphB3 using a chemogenetic approach reversed already-established NP-related behavior in these cervical SCI mice. Furthermore, this phenotypic effect of EphB tyrosine kinase inhibition was sensory modality-specific, affecting mechanical allodynia but not thermal hyperalgesia. Collectively, these findings suggest that enhanced EphB expression and function underlie alterations in excitatory synaptic transmission in the DH and consequent persistent NP following SCI.
28. WHITDRAWN
29. A Neurexin / Neuroligin network underlies sexually dimorphic central synapse formation – SELECTED TALK
Kristen C. Davis and Timothy J. Mosca
30. Olfactory epithelia vs skin fibroblasts; a comparison for neuropsychiatric disorders using a direct conversion paradigm
Msema Msackyi, Wenyu Zhang, Karin Borgmann-Winter and Chang-Gyu Hahn
Most common neuropsychiatric illnesses are complex trait disorders precipitated by many genetic variants and epigenetic modifications. This makes in vitro modeling, important for drug testing and discovery, difficult since the standard for patient-derived in vitro testing of neuropsychiatric disorders are induced pluripotent stem cells which do not retain host tissue epigenetic modifications. This issue is mitigated by directly converting patient-derived tissue into neurons, retaining epigenetics by avoiding a stem cell phase. Epigenetic modifications, which accumulate in response to environmental factors, allow for environmental effects on neuropsychiatric disorders to be measured in an in vitro model. Direct conversion using olfactory epithelia (OE) is desirable for two reasons; neuropsychiatric disorder-associated epigenetic modifications are present in OE, and OE contains neurons potentially shortening the direct conversion timeline. Here we compare skin fibroblasts (SFs), commonly used for direct conversion, and OE in a direct conversion paradigm. We compared these two tissue based on which retains epigenetic modification the best and which has a faster direct conversion rate to neurons. Undifferentiated SFs and OE were compared showing more open chromatin in OE compared to SFs. When the direct conversion rate was compared OE had a higher expression of MAP2 and synapsin proteins at an earlier stage than SFs. Finally, directly converted OE have shown the ability to fire action potentials while SFs have not yet. Taken together, OE could serve as a better source tissue for a direct conversion paradigm compared to SFs, though further experiments are needed for proper determination.
31. An Unbiased Genetic Screen Identifies Axotactin as a Pro-Synaptogenic Factor of Central Nervous System Synapses
Jesse Humenik, Juan Duhart, Timothy J Mosca
32. Tau STED super resolution microscopy enables the study of the nanoscale organization of glutamate receptors in living cells and brain tissue – POSTER AWARD
Rachel E. Cain, Matthew B. Dalva
Synapses are the site of neuronal communication and are essential for brain function, yet studying their organization has been challenging due to their small size. A variety of super-resolution imaging techniques, including Stimulated Emission Depletion (STED) microscopy, have been used to break this barrier. Live-cell STED imaging of over-expressed and fluorescently tagged pre and postsynaptic proteins reveals that synaptic proteins move dynamically in living neurons. However, phototoxicity from STED and the need to express exogenously tagged synaptic proteins limit this approach. To overcome these problems we are developing two new approaches, Tau (τ) STED imaging and CRISPR knock in of fluorescence tags into endogenous glutamate receptors. A gentler form of STED, τSTED can be used to minimize photobleaching, phototoxicity, and background fluorescence. We describe approaches to use a Leica SP8 Tau STED system to image glutamate receptors in tissue and living cultured neurons at high spatial and temporal resolution.
Recently an Open Resource for the Application of Neuronal Genome Editing (ORANGE) has been developed to tag endogenous proteins with fluorescent tags. We describe endogenously tagging glutamate receptors, which can be imaged with live-cell τSTED at a high temporal resolution to visualize the nanoscale dynamics of glutamate receptors in living neurons. Studying the nano-organization of synaptic proteins within brain tissue has been challenging due to the lower resolution that can be achieved in dense tissue. Here, using τSTED imaging in brain sections, we show the capability to study the nanoscale organization of endogenous glutamate receptors in spines in brain tissue.
33. Ephrin-Dependent Transcriptional Regulation Of Extracellular Tyrosine Kinase Vlk
Kolluru D Srikanth , Praveen Chander1, 1, Halley R. Washburn1, Theodore Price , Matthew B Dalva1
The Eph kinases and ephrin partners comprise large family of signaling molecules involved in neural plasticity, development, and disease. The direct interaction between the extracellular domain of EphB2 and GluN1 subunit is mediated by a novel extracellular phosphorylation of a single conserved Y504 on EphB2. Extracellular phosphorylation is underappreciated mechanism but has been found to occur on over 2000 proteins, including many synaptic proteins. We have focused on one kinase, Vertebrate Lonesome Kinase (VLK/PKDCC), a secreted tyrosine-directed kinase that appears localized at synapses. This abstract examines whether EphB2 signaling and neuronal activity can regulate VLK expression. Therefore, we first determined the developmental time course of VLK in rat neurons and mouse brains and found VLK expression decreased with age. Next, whether activation of EphB2 signaling with ephrin-B treatment might induce VLK expression. Interestingly, although the level of VLK constitutive expression decreased with age, ephrin-B treatment increased the expression of VLK only at older ages when baseline expression was lower. VLK expression was also increased by increasing synaptic activity. Next, we asked whether the ephrin-dependent increase in VLK gene expression was NMDAR-dependent. The neurons were treated with ephrin-B in the presence of NMDAR blockers. These data suggest that VLK expression depends only on EphB signaling at DIV14 but requires neuronal activity at DIV21. To understand the mechanism of VLK regulation, we examined the known transcription binding patterns in the VLK promoter and found evidence for STAT3 binding. Consistent with a role for STAT3, inhibition of STAT signaling decreased VLK expression.