The role of kinesin-1 in neuronal dense core vesicle transport, locomotion and lifespan regulation in C. elegans.

Fast axonal transport is crucial for neuronal function and is driven by kinesins and cytoplasmic dynein. Here, we investigated the role of kinesin-1 in dense core vesicle (DCV) transport in C. elegans, using mutants in the kinesin light chains (klc-1 and klc-2) and the motor subunit (unc-116) expressing an ida-1::gfp transgene that labels DCVs. DCV transport in both directions was greatly impaired in an unc-116 mutant and had reduced velocity in a klc-2 mutant. In contrast, the speed of retrograde DCV transport was increased in a klc-1 mutant whereas anterograde transport was unaffected. We identified striking differences between the klc mutants in their effects on worm locomotion and responses to drugs affecting neuromuscular junction activity. We also determined lifespan, finding that unc-116 mutant was short-lived whereas the klc single mutant lifespan was wild type. The ida-1::gfp transgenic strain was also short-lived, but surprisingly, klc-1 and klc-2 extended the ida-1::gfp lifespan beyond that of wild type. Our findings suggest that kinesin-1 not only influences anterograde and retrograde DCV transport but is also involved in regulating lifespan and locomotion, with the two kinesin light chains playing distinct roles.

Age-related upregulation of dense core vesicles in the central inferior colliculus.

Presbycusis is one of the most prevalent disabilities in aged populations of industrialized countries. As we age less excitation reaches the central auditory system from the periphery. To compensate, the central auditory system [e.g., the inferior colliculus (IC)], downregulates GABAergic inhibition to maintain homeostatic balance. However, the continued downregulation of GABA in the IC causes a disruption in temporal precision related to presbycusis. Many studies of age-related changes to neurotransmission in the IC have therefore focused on GABAergic systems. However, we have discovered that dense core vesicles (DCVs) are significantly upregulated with age in the IC. DCVs can carry neuropeptides, co-transmitters, neurotrophic factors, and proteins destined for the presynaptic zone to participate in synaptogenesis. We used immuno transmission electron microscopy across four age groups (3-month; 19-month; 24-month; and 28-month) of Fisher Brown Norway rats to examine the ultrastructure of DCVs in the IC. Tissue was stained post-embedding for GABA immunoreactivity. DCVs were characterized by diameter and by the neurochemical profile (GABAergic/non-GABAergic) of their location (bouton, axon, soma, and dendrite). Our data was collected across the dorsolateral to ventromedial axis of the central IC. After quantification, we had three primary findings. First, the age-related increase of DCVs occurred most robustly in non-GABAergic dendrites in the middle and low frequency regions of the central IC during middle age. Second, the likelihood of a bouton having more than one DCV increased with age. Lastly, although there was an age-related loss of terminals throughout the IC, the proportion of terminals that contained at least one DCV did not decline. We interpret this finding to mean that terminals carrying proteins packaged in DCVs are spared with age. Several recent studies have demonstrated a role for neuropeptides in the IC in defining cell types and regulating inhibitory and excitatory neurotransmission. Given the age-related increase of DCVs in the IC, it will be critical that future studies determine whether (1) specific neuropeptides are altered with age in the IC and (2) if these neuropeptides contribute to the loss of inhibition and/or increase of excitability that occurs during presbycusis and tinnitus.

Activator of KAT3 histone acetyltransferase family ameliorates a neurodevelopmental disorder phenotype in the syntaxin 1A ablated mouse model.

Syntaxin-1A (stx1a) repression causes a neurodevelopmental disorder phenotype, low latent inhibition (LI) behavior, by disrupting 5-hydroxytryptaminergic (5-HTergic) systems. Herein, we discovered that lysine acetyltransferase (KAT) 3B increases stx1a neuronal transcription and TTK21, a KAT3 activator, induces stx1a transcription and 5-HT release in vitro. Furthermore, glucose-derived CSP-TTK21 could restore decreased stx1a expression, 5-HTergic systems in the brain, and low LI in stx1a (+/-) mice by crossing the blood-brain barrier, whereas the KAT3 inhibitor suppresses stx1a expression, 5-HTergic systems, and LI behaviors in wild-type mice. Finally, in wild-type and stx1a (-/-) mice treated with IKK inhibitors and CSP-TTK21, respectively, we show that KAT3 activator-induced LI improvement is a direct consequence of KAT3B-stx1a pathway, not a side effect. In conclusion, KAT3B can positively regulate stx1a transcription in neurons, and increasing neuronal stx1a expression and 5-HTergic systems by a KAT3 activator consequently improves the low LI behavior in the stx1a ablation mouse model.

Pre-fusion motion state determines the heterogeneity of membrane fusion dynamics for large dense-core vesicles.

AIM: In neuroendocrine cells, large dense-core vesicles (LDCVs) undergo highly regulated pre-fusion processes before releasing hormones via membrane fusion. Significant heterogeneity has been found for LDCV population based on the dynamics of membrane fusion. However, how the pre-fusion status impacts the heterogeneity of LDCVs still remains unclear. Hence, we explored pre-fusion determinants of heterogeneous membrane fusion procedure of LDCV subpopulations. METHODS: We assessed the pre-fusion motion of two LDCV subpopulations with distinct membrane fusion dynamics individually, using total internal reflection fluorescence microscopy. These two subpopulations were isolated by blocking Rho GTPase-dependent actin reorganization using Clostridium difficile toxin B (ToxB), which selectively targets the fast fusion vesicle pool. RESULTS: We found that the fast fusion subpopulation was in an active motion mode prior to release, termed "active" LDCV pool, while vesicles from the slow fusion subpopulation were also moving but in a significantly more confined status, forming an "inert" pool. The depletion of the active pool by ToxB also eliminated fast fusion vesicles and was not rescued by pre-treatment with phorbol ester. A mild actin reorganization blocker, latrunculin A, that partially disrupted the active pool, only slightly attenuated the fast fusion subpopulation. CONCLUSION: The pre-fusion motion state of LDCVs also exhibits heterogeneity and dictates the heterogeneous fusion pore dynamics. Rearrangement of F-actin network mediates vesicle pre-fusion motion and subsequently determines the membrane fusion kinetics.

Endoplasmic reticulum stress impedes regulated secretion by governing key exocytotic and granulogenic molecular switches.

Dense core vesicles (DCVs) and synaptic vesicles are specialised secretory vesicles in neurons and neuroendocrine cells, and abnormal release of their cargo is associated with various pathophysiologies. Endoplasmic reticulum (ER) stress and inter-organellar communication are also associated with disease biology. To investigate the functional status of regulated exocytosis arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using thapsigargin. DCV exocytosis was severely compromised in ER-stressed PC12 cells and was reversed to varying magnitudes by ER stress attenuators. Experiments with tunicamycin, an independent ER stressor, yielded similar results. Concurrently, ER stress also caused impaired DCV exocytosis in insulin-secreting INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4, an inhibitor of CREB that binds to the CREB-binding site. The effects of loss of function of ATF4 in ER-stressed cells substantiated this attribution. Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key exocytotic and granulogenic switches regulated via the eIF2α (EIF2A)-ATF4 axis.

Amperometry and Electron Microscopy show Stress Granules Induce Homotypic Fusion of Catecholamine Vesicles.

An overreactive stress granule (SG) pathway and long-lived, stable SGs formation are thought to participate in the progress of neurodegenerative diseases (NDs). To understand if and how SGs contribute to disorders of neurotransmitter release in NDs, we examined the interaction between extracellular isolated SGs and vesicles. Amperometry shows that the vesicular content increases and dynamics of vesicle opening slow down after vesicles are treated with SGs, suggesting larger vesicles are formed. Data from transmission electron microscopy (TEM) clearly shows that a portion of large dense-core vesicles (LDCVs) with double/multiple cores appear, thus confirming that SGs induce homotypic fusion between LDCVs. This might be a protective step to help cells to survive following high oxidative stress. A hypothetical mechanism is proposed whereby enriched mRNA or protein in the shell of SGs is likely to bind intrinsically disordered protein (IDP) regions of vesicle associated membrane protein (VAMP) driving a disrupted membrane between two closely buddled vesicles to fuse with each other to form double-core vesicles. Our results show that SGs induce homotypic fusion of LDCVs, providing better understanding of how SGs intervene in pathological processes and opening a new direction to investigations of SGs involved neurodegenerative disease.

Distinct Alterations in Dendritic Spine Morphology in the Absence of ß-Neurexins.

Dendritic spines are essential for synaptic function because they constitute the postsynaptic compartment of the neurons that receives the most excitatory input. The extracellularly shorter variant of the presynaptic cell adhesion molecules neurexins, ß-neurexin, has been implicated in various aspects of synaptic function, including neurotransmitter release. However, its role in developing or stabilizing dendritic spines as fundamental computational units of excitatory synapses has remained unclear. Here, we show through morphological analysis that the deletion of ß-neurexins in hippocampal neurons in vitro and in hippocampal tissue in vivo affects presynaptic dense-core vesicles, as hypothesized earlier, and, unexpectedly, alters the postsynaptic spine structure. Specifically, we observed that the absence of ß-neurexins led to an increase in filopodial-like protrusions in vitro and more mature mushroom-type spines in the CA1 region of adult knockout mice. In addition, the deletion of ß-neurexins caused alterations in the spine head dimension and an increase in spines with perforations of their postsynaptic density but no changes in the overall number of spines or synapses. Our results indicate that presynaptic ß-neurexins play a role across the synaptic cleft, possibly by aligning with postsynaptic binding partners and glutamate receptors via transsynaptic columns.

A vesicular Warburg effect: Aerobic glycolysis occurs on axonal vesicles for local NAD+ recycling and transport.

In neurons, fast axonal transport (FAT) of vesicles occurs over long distances and requires constant and local energy supply for molecular motors in the form of adenosine triphosphate (ATP). FAT is independent of mitochondrial metabolism. Indeed, the glycolytic machinery is present on vesicles and locally produces ATP, as well as nicotinamide adenine dinucleotide bonded with hydrogen (NADH) and pyruvate, using glucose as a substrate. It remains unclear whether pyruvate is transferred to mitochondria from the vesicles as well as how NADH is recycled into NAD+ on vesicles for continuous glycolysis activity. The optimization of a glycolytic activity test for subcellular compartments allowed the evaluation of the kinetics of vesicular glycolysis in the brain. This revealed that glycolysis is more efficient on vesicles than in the cytosol. We also found that lactate dehydrogenase (LDH) enzymatic activity is required for effective vesicular ATP production. Indeed, inhibition of LDH or the forced degradation of pyruvate inhibited ATP production from axonal vesicles. We found LDHA rather than the B isoform to be enriched on axonal vesicles suggesting a preferential transformation of pyruvate to lactate and a concomitant recycling of NADH into NAD+ on vesicles. Finally, we found that LDHA inhibition dramatically reduces the FAT of both dense-core vesicles and synaptic vesicle precursors in a reconstituted cortico-striatal circuit on-a-chip. Together, this shows that aerobic glycolysis is required to supply energy for vesicular transport in neurons, similar to the Warburg effect.

Maximal Fusion Capacity and Efficient Replenishment of the Dense Core Vesicle Pool in Hippocampal Neurons.

Neuropeptides and neurotrophins, stored in dense core vesicles (DCVs), are together the largest currently known group of chemical signals in the brain. Exocytosis of DCVs requires high-frequency or patterned stimulation, but the determinants to reach maximal fusion capacity and for efficient replenishment of released DCVs are unknown. Here, we systematically studied fusion of DCV with single vesicle resolution on different stimulation patterns in mammalian CNS neurons. We show that tetanic stimulation trains of 50-Hz action potential (AP) bursts maximized DCV fusion, with significantly fewer fusion event during later bursts of the train. This difference was omitted by introduction of interburst intervals but did not increase total DCV fusion. Interburst intervals as short as 5 s were sufficient to restore the fusion capacity. Theta burst stimulation (TBS) triggered less DCV fusion than tetanic stimulation, but a similar fusion efficiency per AP. Prepulse stimulation did not alter this. However, low-frequency stimulation (4 Hz) intermitted with fast ripple stimulation (200 APs at 200 Hz) produced substantial DCV fusion, albeit not as much as tetanic stimulation. Finally, individual fusion events had longer durations with more intense stimulation. These data indicate that trains of 50-Hz AP stimulation patterns triggered DCV exocytosis most efficiently and more intense stimulation promotes longer DCV fusion pore openings.SIGNIFICANCE STATEMENT Neuropeptides and neurotrophins modulate multiple regulatory functions of human body like reproduction, food intake or mood. They are packed into dense core vesicles (DCVs) that undergo calcium and action potential (AP) fusion with the plasma membrane. In order to study the fusion of DCVs in vitro, techniques like perfusion with buffer containing high concentration of potassium or electric field stimulation are needed to trigger the exocytosis of DCVs. Here, we studied the relationship between DCVs fusion properties and different electric field stimulation patterns. We used six different stimulation patterns and showed that trains of 50-Hz action potential bursts triggered DCV exocytosis most efficiently and more intense stimulation promotes longer DCV fusion pore openings.

Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons.

Tomosyn is a large, non-canonical SNARE protein proposed to act as an inhibitor of SNARE complex formation in the exocytosis of secretory vesicles. In the brain, tomosyn inhibits the fusion of synaptic vesicles (SVs), whereas its role in the fusion of neuropeptide-containing dense core vesicles (DCVs) is unknown. Here, we addressed this question using a new mouse model with a conditional deletion of tomosyn (Stxbp5) and its paralogue tomosyn-2 (Stxbp5l). We monitored DCV exocytosis at single vesicle resolution in tomosyn-deficient primary neurons using a validated pHluorin-based assay. Surprisingly, loss of tomosyns did not affect the number of DCV fusion events but resulted in a strong reduction of intracellular levels of DCV cargos, such as neuropeptide Y (NPY) and brain-derived neurotrophic factor (BDNF). BDNF levels were largely restored by re-expression of tomosyn but not by inhibition of lysosomal proteolysis. Tomosyn's SNARE domain was dispensable for the rescue. The size of the trans-Golgi network and DCVs was decreased, and the speed of DCV cargo flux through Golgi was increased in tomosyn-deficient neurons, suggesting a role for tomosyns in DCV biogenesis. Additionally, tomosyn-deficient neurons showed impaired mRNA expression of some DCV cargos, which was not restored by re-expression of tomosyn and was also observed in Cre-expressing wild-type neurons not carrying loxP sites, suggesting a direct effect of Cre recombinase on neuronal transcription. Taken together, our findings argue against an inhibitory role of tomosyns in neuronal DCV exocytosis and suggests an evolutionary conserved function of tomosyns in the packaging of secretory cargo at the Golgi.

Functional Evidence of CCDC186 as a New Disease-Associated Gene with Endocrine and Central Nervous System Alterations.

CCDC186 protein is involved in the maturation of dense-core vesicles (DCVs) in the trans-Golgi network in neurons and endocrine cells. Mutations in genes involved in DCV regulation, other than CCDC186, have been described in patients with neurodevelopmental disorders. To date, only one patient, within a large sequencing study of 1000 cases, and a single case report with variants in CCDC186, had previously been described. However, no functional studies in any of these two cases had been performed. We identified three patients from two gypsy families, unrelated to each other, with mutations in the CCDC186 gene. Clinically, all patients presented with seizures, frontotemporal atrophy, hypomyelination, recurrent infections, and endocrine disturbances such as severe non-ketotic hypoglycemia. Low levels of cortisol, insulin, or growth hormone could only be verified in one patient. All of them had a neonatal onset and died between 7 months and 4 years of age. Whole exome sequencing identified a homozygous variant in the CCDC186 gene (c.2215C>T, p.Arg739Ter) in the index patients of both families. Protein expression studies demonstrated that CCDC186 was almost undetectable in fibroblasts and muscle tissue. These observations correlated with the transcriptomic analysis performed in fibroblasts in one of the patients, which showed a significant reduction of CCDC186 mRNA levels. Our study provides functional evidence that mutations in this gene have a pathogenic effect on the protein and reinforces CCDC186 as a new disease-associated gene. In addition, mutations in CCDC186 could explain the combined endocrine and neurologic alterations detected in our patients.

Exploiting Microelectrode Geometry for Comprehensive Detection of Individual Exocytosis Events at Single Cells.

Carbon fiber microelectrodes are commonly used for real-time monitoring of individual exocytosis events at single cells. Since the nature of an electrochemical signal is fundamentally governed by mass transport to the electrode surface, microelectrode geometry can be exploited to achieve precise and accurate measurements. Researchers traditionally pair amperometric measurements of exocytosis with a ∼10-µm diameter, disk microelectrode in an "artificial synapse" configuration to directly monitor individual release events from single cells. Exocytosis is triggered, and released molecules diffuse to the "post-synaptic" electrode for oxidation. This results in a series of distinct current spikes corresponding to individual exocytosis events. However, it remains unclear how much of the material escapes detection. In this work, the performance of 10- and 34-µm diameter carbon fiber disk microelectrodes was directly compared in monitoring exocytosis at single chromaffin cells. The 34-µm diameter electrode was more sensitive to catecholamines and enkephalins than its traditional, 10-µm diameter counterpart, and it more effectively covered the entire cell. As such, the larger sensor detected more exocytosis events overall, as well as a larger quantal size, suggesting that the traditional tools underestimate the above measurements. Both sensors reliably measured l-DOPA-evoked changes in quantal size, and both exhibited diffusional loss upon adjustment of cell-electrode spacing. Finite element simulations using COMSOL support the improved collection efficiency observed using the larger sensor. Overall, this work demonstrates how electrode geometry can be exploited for improved detection of exocytosis events by addressing diffusional loss─an often-overlooked source of inaccuracy in single-cell measurements.

Damaged mitochondria coincide with presynaptic vesicle loss and abnormalities in alzheimer's disease brain.

Loss of synapses is the most robust pathological correlate of Alzheimer's disease (AD)-associated cognitive deficits, although the underlying mechanism remains incompletely understood. Synaptic terminals have abundant mitochondria which play an indispensable role in synaptic function through ATP provision and calcium buffering. Mitochondrial dysfunction is an early and prominent feature in AD which could contribute to synaptic deficits. Here, using electron microscopy, we examined synapses with a focus on mitochondrial deficits in presynaptic axonal terminals and dendritic spines in cortical biopsy samples from clinically diagnosed AD and age-matched non-AD control patients. Synaptic vesicle density within the presynaptic axon terminals was significantly decreased in AD cases which appeared largely due to significantly decreased reserve pool, but there were significantly more presynaptic axons containing enlarged synaptic vesicles or dense core vesicles in AD. Importantly, there was reduced number of mitochondria along with significantly increased damaged mitochondria in the presynapse of AD which correlated with changes in SV density. Mitochondria in the post-synaptic dendritic spines were also enlarged and damaged in the AD biopsy samples. This study provided evidence of presynaptic vesicle loss as synaptic deficits in AD and suggested that mitochondrial dysfunction in both pre- and post-synaptic compartments contribute to synaptic deficits in AD.

Molecular heterogeneity of C. elegans glia across sexes.

A comprehensive description of nervous system function, and sex dimorphism within, is incomplete without clear assessment of the diversity of its component cell types, neurons and glia. C. elegans has an invariant nervous system with the first mapped connectome of a multicellular organism and single-cell atlas of component neurons. Here we present single nuclear RNA-seq evaluation of glia across the entire adult C. elegans nervous system, including both sexes. Machine learning models enabled us to identify both sex-shared and sex-specific glia and glial subclasses. We have identified and validated molecular markers in silico and in vivo for these molecular subcategories. Comparative analytics also reveals previously unappreciated molecular heterogeneity in anatomically identical glia between and within sexes, indicating consequent functional heterogeneity. Furthermore, our datasets reveal that while adult C. elegans glia express neuropeptide genes, they lack the canonical unc-31/CAPS-dependent dense core vesicle release machinery. Thus, glia employ alternate neuromodulator processing mechanisms. Overall, this molecular atlas, available at www.wormglia.org, reveals rich insights into heterogeneity and sex dimorphism in glia across the entire nervous system of an adult animal.

Rat Pheochromocytoma PC12 Cells in Culture.

PC12 cells serve as a secretory cell model, especially suitable for studying the molecular mechanisms underlying fusion pore kinetics in regulated exocytosis of dense-core vesicles (DCVs). In this chapter, we describe a series of PC12 cell culture procedures optimized for real-time functional assays such as single-vesicle amperometry. In addition, these conditions have been widely used for single-cell biochemical assays such as the proximity ligation assay with immunostaining.

Isolation and Purification of Chromaffin Granules from Adrenal Glands and Cultured Neuroendocrine Cells.

Chromaffin granules isolated from adrenal glands constitute a powerful experimental tool to the study of secretory vesicle components and their participation in fusion and docking processes, vesicle aggregation, and interactions with cytosolic components. Although it is possible to isolate and purify chromaffin granules from adrenal glands of different species, bovine adrenal glands are the most used tissue source due to its easy handling and the large amount of granules that can be obtained from this tissue. In this chapter, we describe an easy-to-use and short-term protocol for efficiently obtaining highly purified chromaffin granules from bovine adrenal medulla. We additionally include protocols to isolate granules from cultured bovine chromaffin cells and PC12 cells, as well as a section to obtain chromaffin granules from mouse adrenal glands.

High-speed imaging reveals the bimodal nature of dense core vesicle exocytosis.

During exocytosis, the fusion of secretory vesicle with plasma membrane forms a pore that regulates release of neurotransmitter and peptide. Heterogeneity of fusion pore behavior has been attributed to stochastic variation in a common exocytic mechanism, implying a lack of biological control. Using a fluorescent false neurotransmitter (FFN), we imaged dense core vesicle (DCV) exocytosis in primary mouse adrenal chromaffin cells by total internal reflection fluorescence microscopy at millisecond resolution and observed strikingly divergent modes of release, with fast events lasting <30 ms and slow events persisting for seconds. Dual imaging of slow events shows a delay in the entry of external dye relative to FFN release, suggesting exclusion by an extremely narrow pore <1 nm in diameter. Unbiased comprehensive analysis shows that the observed variation cannot be explained by stochasticity alone, but rather involves distinct mechanisms, revealing the bimodal nature of DCV exocytosis. Further, loss of calcium sensor synaptotagmin 7 increases the proportion of slow events without changing the intrinsic properties of either class, indicating the potential for independent regulation. The identification of two distinct mechanisms for release capable of independent regulation suggests a biological basis for the diversity of fusion pore behavior.

Differential axonal trafficking of Neuropeptide Y-, LAMP1-, and RAB7-tagged organelles in vivo.

Different organelles traveling through neurons exhibit distinct properties in vitro, but this has not been investigated in the intact mammalian brain. We established simultaneous dual color two-photon microscopy to visualize the trafficking of Neuropeptide Y (NPY)-, LAMP1-, and RAB7-tagged organelles in thalamocortical axons imaged in mouse cortex in vivo. This revealed that LAMP1- and RAB7-tagged organelles move significantly faster than NPY-tagged organelles in both anterograde and retrograde direction. NPY traveled more selectively in anterograde direction than LAMP1 and RAB7. By using a synapse marker and a calcium sensor, we further investigated the transport dynamics of NPY-tagged organelles. We found that these organelles slow down and pause at synapses. In contrast to previous in vitro studies, a significant increase of transport speed was observed after spontaneous activity and elevated calcium levels in vivo as well as electrically stimulated activity in acute brain slices. Together, we show a remarkable diversity in speeds and properties of three axonal organelle marker in vivo that differ from properties previously observed in vitro.

Estrogen differentially regulates transcriptional landscapes of preoptic and arcuate kisspeptin neuron populations.

Kisspeptin neurons residing in the rostral periventricular area of the third ventricle (KPRP3V) and the arcuate nucleus (KPARC) mediate positive and negative estrogen feedback, respectively. Here, we aim to compare transcriptional responses of KPRP3V and KPARC neurons to estrogen. Transgenic mice were ovariectomized and supplemented with either 17ß-estradiol (E2) or vehicle. Fluorescently tagged KPRP3V neurons collected by laser-capture microdissection were subjected to RNA-seq. Bioinformatics identified 222 E2-dependent genes. Four genes encoding neuropeptide precursors (Nmb, Kiss1, Nts, Penk) were robustly, and Cartpt was subsignificantly upregulated, suggesting putative contribution of multiple neuropeptides to estrogen feedback mechanisms. Using overrepresentation analysis, the most affected KEGG pathways were neuroactive ligand-receptor interaction and dopaminergic synapse. Next, we re-analyzed our previously obtained KPARC neuron RNA-seq data from the same animals using identical bioinformatic criteria. The identified 1583 E2-induced changes included suppression of many neuropeptide precursors, granins, protein processing enzymes, and other genes related to the secretory pathway. In addition to distinct regulatory responses, KPRP3V and KPARC neurons exhibited sixty-two common changes in genes encoding three hormone receptors (Ghsr, Pgr, Npr2), GAD-65 (Gad2), calmodulin and its regulator (Calm1, Pcp4), among others. Thirty-four oppositely regulated genes (Kiss1, Vgf, Chrna7, Tmem35a) were also identified. The strikingly different transcriptional responses in the two neuron populations prompted us to explore the transcriptional mechanism further. We identified ten E2-dependent transcription factors in KPRP3V and seventy in KPARC neurons. While none of the ten transcription factors interacted with estrogen receptor-α, eight of the seventy did. We propose that an intricate, multi-layered transcriptional mechanism exists in KPARC neurons and a less complex one in KPRP3V neurons. These results shed new light on the complexity of estrogen-dependent regulatory mechanisms acting in the two functionally distinct kisspeptin neuron populations and implicate additional neuropeptides and mechanisms in estrogen feedback.

Defective axonal transport of endo-lysosomes and dense core vesicles in a Drosophila model of C9-ALS/FTD.

A GGGGCC (G4 C2 ) repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although disruptions in axonal transport are implicated in the pathogenesis of multiple neurodegenerative diseases, the underlying mechanisms causing these defects remain unclear. Here, we performed live imaging of Drosophila motor neurons expressing expanded G4 C2 repeats in third-instar larvae and investigated the axonal transport of multiple organelles in vivo. Expression of expanded G4 C2 repeats causes an increase in static axonal lysosomes, while it impairs trafficking of late endosomes (LEs) and dense core vesicles (DCVs). Surprisingly, however, axonal transport of mitochondria is unaffected in motor axons expressing expanded G4 C2 repeats. Thus, our data indicate that expanded G4 C2 repeat expression differentially impacts axonal transport of vesicular organelles and mitochondria in Drosophila models of C9orf72-associated ALS/FTD.