Dendritic Morphology Of Neurons Research Articles

Sumoylation in astrocytes induces changes in the proteome of the derived small extracellular vesicles which change protein synthesis and dendrite morphology in target neurons

Emerging evidence highlights the relevance of the protein post-translational modification by SUMO (Small Ubiquitin-like Modifier) in the central nervous system for modulating cognition and plasticity in health and disease. In these processes, astrocyte-to-neuron crosstalk mediated by extracellular vesicles (EVs) plays a yet poorly understood role. Small EVs (sEVs), including microvesicles and exosomes, contain a molecular cargo of lipids, proteins, and nucleic acids that define their biological effect on target cells. Here, we investigated whether SUMOylation globally impacts the sEV protein cargo. For this, sEVs were isolated from primary cultures of astrocytes by ultracentrifugation or using a commercial sEV isolation kit. SUMO levels were regulated: 1) via plasmids that over-express SUMO, or 2) via experimental conditions that increase SUMOylation, i.e., by using the stress hormone corticosterone, or 3) via the SUMOylation inhibitor 2-D08 (2′,3′,4′-trihydroxy-flavone, 2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one). Corticosterone and 2-D08 had opposing effects on the number of sEVs and on their protein cargo. Proteomic analysis showed that increased SUMOylation in corticosterone-treated or plasmid-transfected astrocytes increased the presence of proteins related to cell division, transcription, and protein translation in the derived sEVs. When sEVs derived from corticosterone-treated astrocytes were transferred to neurons to assess their impact on protein synthesis using the fluorescence non-canonical amino acid tagging assay (FUNCAT), we detected an increase in protein synthesis, while sEVs from 2-D08-treated astrocytes had no effect. Our results show that SUMO conjugation plays an important role in the modulation of the proteome of astrocyte-derived sEVs with a potential functional impact on neurons.

Short-term Dendritic Dynamics of Neonatal Cortical Neurons Revealed by in vivo Imaging with Improved Spatiotemporal Resolution.

Individual neurons in sensory cortices exhibit specific receptive fields based on their dendritic patterns. These dendritic morphologies are established and refined during the neonatal period through activity-dependent plasticity. This process can be visualized using two-photon in vivo time-lapse imaging, but sufficient spatiotemporal resolution is essential. We previously examined dendritic patterning from spiny stellate (SS) neurons, the major type of layer 4 (L4) neurons, in the mouse primary somatosensory cortex (barrel cortex), where mature dendrites display a strong orientation bias toward the barrel center. Longitudinal imaging at 8-h intervals revealed the long-term dynamics by which SS neurons acquire this unique dendritic pattern. However, the spatiotemporal resolution was insufficient to detect the more rapid changes in SS neuron dendrite morphology during the critical neonatal period. In the current study, we imaged neonatal L4 neurons hourly for 8 h and improved the spatial resolution by uniform cell-surface labeling. The improved spatiotemporal resolution allowed detection of precise changes in dendrite morphology and revealed aspects of short-term dendritic dynamics unique to the neonatal period. Basal dendrites of barrel cortex L4 neurons were highly dynamic. In particular, both barrel-inner and barrel-outer dendrites (trees and branches) emerged/elongated and disappeared/retracted at similarly high frequencies, suggesting that SS neurons acquire biased dendrite patterns through rapid trial-and-error emergence, elongation, elimination, and retraction of dendritic trees and branches. We also found correlations between morphology and behavior (elongation/retraction) of dendritic tips. Thus, the current study revealed short-term dynamics and related features of cortical neuron dendrites during refinement.Significance StatementThe formation of proper dendritic patterns during early postnatal development is essential for normal neuronal circuit function in adulthood. To elucidate the mechanisms responsible for this refinement, in vivo imaging with high spatiotemporal resolution is useful. Our previous long-term in vivo imaging studies have clarified aspects of dendritic refinement mechanism; however, due to the long intervals (8-h) between image acquisitions, rapid changes in dendritic morphology were missed. Here hourly in vivo time-lapse imaging of neonatal mouse barrel cortex over 8 h revealed the rapid changes in the dendrite morphology of layer 4 neurons, thereby providing a more comprehensive record of dendritic refinement during postnatal development for mechanistic analysis.

Progressive inflammation reduces high-frequency EEG activity and cortical dendritic arborisation in late gestation fetal sheep

BackgroundAntenatal infection/inflammation is associated with disturbances in neuronal connectivity, impaired cortical growth and poor neurodevelopmental outcomes. The pathophysiological substrate that underpins these changes is poorly understood. We tested the hypothesis that progressive inflammation in late gestation fetal sheep would alter cortical neuronal microstructure and neural function assessed using electroencephalogram band power analysis.MethodsFetal sheep (0.85 of gestation) were surgically instrumented for continuous electroencephalogram (EEG) recording and randomly assigned to repeated saline (control; n = 9) or LPS (0 h = 300 ng, 24 h = 600 ng, 48 h = 1200 ng; n = 8) infusions to induce inflammation. Sheep were euthanised 4 days after the first LPS infusion for assessment of inflammatory gene expression, histopathology and neuronal dendritic morphology in the somatosensory cortex.ResultsLPS infusions increased delta power between 8 and 50 h, with reduced beta power from 18 to 96 h (P < 0.05 vs. control). Basal dendritic length, numbers of dendritic terminals, dendritic arborisation and numbers of dendritic spines were reduced in LPS-exposed fetuses (P < 0.05 vs. control) within the somatosensory cortex. Numbers of microglia and interleukin (IL)-1β immunoreactivity were increased in LPS-exposed fetuses compared with controls (P < 0.05). There were no differences in total numbers of cortical NeuN + neurons or cortical area between the groups.ConclusionsExposure to antenatal infection/inflammation was associated with impaired dendritic arborisation, spine number and loss of high-frequency EEG activity, despite normal numbers of neurons, that may contribute to disturbed cortical development and connectivity.

P120-catenin subfamily members have distinct as well as shared effects on dendrite morphology during neuron development in vitro.

Dendritic arborization is essential for proper neuronal connectivity and function. Conversely, abnormal dendrite morphology is associated with several neurological pathologies like Alzheimer's disease and schizophrenia. Among major intrinsic mechanisms that determine the extent of the dendritic arbor is cytoskeletal remodeling. Here, we characterize and compare the impact of the four proteins involved in cytoskeletal remodeling-vertebrate members of the p120-catenin subfamily-on neuronal dendrite morphology. In relation to each of their own distributions, we find that p120-catenin and delta-catenin are expressed at relatively higher proportions in growth cones compared to ARVCF-catenin and p0071-catenin; ARVCF-catenin is expressed at relatively high proportions in the nucleus; and all catenins are expressed in dendritic processes and the soma. Through altering the expression of each p120-subfamily catenin in neurons, we find that exogenous expression of either p120-catenin or delta-catenin correlates with increased dendritic length and branching, whereas their respective depletion decreases dendritic length and branching. While increasing ARVCF-catenin expression also increases dendritic length and branching, decreasing expression has no grossly observable morphological effect. Finally, increasing p0071-catenin expression increases dendritic branching, but not length, while decreasing expression decreases dendritic length and branching. These distinct localization patterns and morphological effects during neuron development suggest that these catenins have both shared and distinct roles in the context of dendrite morphogenesis.

Delayed administration of Trichilia catigua A. Juss. Ethyl-acetate fraction after cerebral ischemia prevents spatial memory deficits, decreases oxidative stress, and impacts neural plasticity in rats.

Trichilia catigua A. Juss (Meliaceae) is used in Brazilian folk medicine to alleviate fatigue and emotional stress and improve memory. Previous studies from our laboratory reported that an ethyl-acetate fraction (EAF) of T. catigua that was given before cerebral ischemia in vivo prevented memory loss and reduced oxidative stress and neuroinflammation. Despite the value of these findings of a neuroprotective effect of T. catigua, treatment that was given immediately before or immediately after ischemia limits its clinical relevance. Thus, unknown is whether T. catigua possesses a specific time window of efficacy (TWE) when administered postischemia. Given continuity to previous studies, we investigated whether an EAF of T. catigua maintains its neuroprotective properties if treatment begins at different time windows of efficacy after ischemia. We also evaluated, for the first time, whether T. catigua possesses neuroplasticity/neurotrophic properties. Rats were subjected to transient global brain ischemia (TGCI) and then given a single dose of the EAF (400mg/kg) or vehicle (1ml/kg) orally 1, 4, or 6h postischemia. The levels of protein PCG, GSH, and GSSG, and activity of SOD and CAT were assayed as markers of oxidative stress on the day after ischemia. In another experiment, naive rats underwent spatial learning training in a radial maze task and then subjected to TGCI. Delayed treatment with the EAF began 4 or 6h later and continued for 7 days. Retrograde memory performance was assessed 10, 17, and 24 days postischemia. Afterward, brains were examined for neurodegeneration and neuronal dendritic morphology in the hippocampus and cerebral cortex. Another group received the EAF at 4h of reperfusion, and 4 days later their brains were examined for GFAP and Iba-1 immunoreactivity. Lastly, ischemic rats received the EAF 4h after ischemia and neural plasticity-related proteins, BDNF, SYN, PSD 95, and NeuN were measured in the hippocampus 7 and 14 days after ischemia. A single EAF administration 1, 4, or 6h postischemia alleviated oxidative stress that was caused by ischemia, expressed as a reduction of the amount of the PCG and GSSG, normalization of the GSH/GSSG ratio, and the restoration of SOD activity. Ischemia caused the persistent loss of memory (i.e., amnesia), an outcome that was consistently ameliorated by treatment with the EAF that was initiated 4 or 6h postischemia. The 4h delay in EAF treatment positively impacted dendritic morphology in neurons that survived ischemia. TGCI reduced BDNF, SYN, PSD-95, and NeuN protein levels in the hippocampus and cerebral cortex. The EAF normalized SYN and PSD-95 protein levels. Ischemia-induced neurodegeneration and glial cell activation were not prevented by EAF treatment. The present study corroborates prior data that demonstrated the neuroprotective potential of T. catigua and extends these data by showing that the delayed administration of EAF postischemia effectively prevented memory impairment and decreased oxidative stress, dendritic deterioration, and synaptic protein loss within a TWE that ranged from 1 to 6h. This specific TWE in preclinical research may have clinical relevance by suggesting the possible utility of this plant for the development of neuroprotective strategies in the setting of ischemic brain diseases. Another innovative finding of the present study was the possible neurotrophic/neuroplastic properties of T. catigua.

Morphological changes of large layer V pyramidal neurons in cortical motor-related areas after spinal cord injury in macaque monkeys

In primates, neurons giving rise to the corticospinal tract (CST) are distributed in several motor-related areas of the frontal lobe, such as the primary motor cortex (M1), the supplementary motor area (SMA), and the dorsal and ventral divisions of the premotor cortex (PMd, PMv). Recently, we have shown in macaque monkeys that the morphology of basal dendrites of CST neurons, i.e., large layer V pyramidal neurons, varies among the digit regions of the motor-related areas. Here, we investigated the alterations in basal dendrite morphology of CST neurons after spinal cord injury (SCI). In our monkey model, both the complexity and the spine density of basal dendrites were highly decreased throughout the areas. Notably, these events were less prominent for the PMd than for the M1, SMA, and PMv. In analyzing the density changes post-SCI of the filopodia-, thin-, stubby-, and mushroom-type spines, it was found that the density of filopodia-type spines was increased for all areas, whereas the other types of spines exhibited density decreases. Such spine density reductions were so limited for the PMd as compared to the other areas. The observed plastic changes of CST neurons may contribute to the recovery from impaired motor functions caused by SCI.

Induction of Bdnf from promoter I following electroconvulsive seizures contributes to structural plasticity in neurons of the piriform cortex

BackgroundElectroconvulsive therapy (ECT) efficacy is hypothesized to depend on induction of molecular and cellular events that trigger neuronal plasticity. Investigating how electroconvulsive seizures (ECS) impact plasticity in animal models can help inform our understanding of basic mechanisms by which ECT relieves symptoms of depression. ECS-induced plasticity is associated with differential expression of unique isoforms encoding the neurotrophin, brain-derived neurotrophic factor (BDNF). HypothesisWe hypothesized that cells expressing the Bdnf exon 1-containing isoform are important for ECS-induced structural plasticity in the piriform cortex, a highly epileptogenic region that is responsive to ECS. MethodsWe selectively labeled Bdnf exon 1-expressing neurons in mouse piriform cortex using Cre recombinase dependent on GFP technology (CRE-DOG). We then quantified changes in dendrite morphology and density of Bdnf exon 1-expressing neurons. ResultsLoss of promoter I-derived BDNF caused changes in spine density and morphology in Bdnf exon 1-expressing neurons following ECS. ConclusionsPromoter I-derived Bdnf is required for ECS-induced dendritic structural plasticity in Bdnf exon 1-expressing neurons.

Carboxypeptidase E Independently Changes Microtubule Glutamylation, Dendritic Branching, and Neuronal Migration.

Neuronal migration and dendritogenesis are dependent on dynamic changes to the microtubule (MT) network. Among various factors that regulate MT dynamics and stability, post-translational modifications (PTMs) of MTs play a critical role in conferring specificity of regulatory protein binding to MTs. Thus, it is important to understand the regulation of PTMs during brain development as multiple developmental processes are dependent on MTs. In this study, we identified that carboxypeptidase E (CPE) changes tubulin polyglutamylation, a major PTM in the brain, and we examine the impact of CPE-mediated changes to polyglutamylation on cortical neuron migration and dendrite morphology. We show, for the first time, that overexpression of CPE increases the level of polyglutamylated α-tubulin while knockdown decreases the level of polyglutamylation. We also demonstrate that CPE-mediated changes to polyglutamylation are dependent on the CPE zinc-binding motif and that this motif is necessary for CPE action on p150Glued localization. However, overexpression of a CPE mutant that does not increase MT glutamylation mimics the effects of overexpression of wild type CPE on dendrite branching. Furthermore, although overexpression of wild type CPE does not alter cortical neuron migration, overexpression of the mutant may act in a dominant-negative manner as it decreases the number of neurons that reach the cortical plate (CP), as we previously reported for CPE knockdown. Overall, our data suggest that CPE changes MT glutamylation and redistribution of p150Glued and that this function of CPE is independent of its role in shaping dendrite development but plays a partial role in regulating cortical neuron migration.

Sustained Hippocampal Neural Plasticity Questions the Reproducibility of an Amyloid-β-Induced Alzheimer's Disease Model.

The use of Alzheimer's disease (AD) models obtained by intracerebral infusion of amyloid-β (Aβ) has been increasingly reported in recent years. Nonetheless, these models may present important challenges. We have focused on canonical mechanisms of hippocampal-related neural plasticity to characterize a rat model obtained by an intracerebroventricular (icv) injection of soluble amyloid-β42 (Aβ42). Animal behavior was evaluated in the elevated plus maze, Y-Maze spontaneous or forced alternation, Morris water maze, and open field, starting 2 weeks post-Aβ42 infusion. Hippocampal neurogenesis was assessed 3 weeks after Aβ42 injection. Aβ deposition, tropomyosin receptor kinase B levels, and neuroinflammation were appraised at 3 and 14 days post-Aβ42 administration. We found that immature neuronal dendritic morphology was abnormally enhanced, but proliferation and neuronal differentiation in the dentate gyrus was conserved one month after Aβ42 injection. Surprisingly, animal behavior did not reveal changes in cognitive performance nor in locomotor and anxious-related activity. Brain-derived neurotrophic factor related-signaling was also unchanged at 3 and 14 days post-Aβ icv injection. Likewise, astrocytic and microglial markers of neuroinflammation in the hippocampus were unaltered in these time points. Taken together, our data emphasize a high variability and lack of behavioral reproducibility associated with these Aβ injection-based models, as well as the need for its further optimization, aiming at addressing the gap between preclinical AD models and the human disorder.

HoxD transcription factors define monosynaptic sensory-motor specificity in the developing spinal cord.

The specificity of monosynaptic connections between proprioceptive sensory neurons and their recipient spinal motor neurons depends on multiple factors, including motor neuron positioning and dendrite morphology, axon projection patterns of proprioceptive sensory neurons in the spinal cord, and the ligand-receptor molecules involved in cell-to-cell recognition. However, with few exceptions, the transcription factors engaged in this process are poorly characterized. Here, we show that members of the HoxD family of transcription factors play a crucial role in the specificity of monosynaptic sensory-motor connections. Mice lacking Hoxd9, Hoxd10 and Hoxd11 exhibit defects in locomotion but have no obvious defects in motor neuron positioning or dendrite morphology through the medio-lateral and rostro-caudal axes. However, we found that quadriceps motor neurons in these mice show aberrant axon development and receive inappropriate inputs from proprioceptive sensory axons innervating the obturator muscle. These genetic studies demonstrate that the HoxD transcription factors play an integral role in the synaptic specificity of monosynaptic sensory-motor connections in the developing spinal cord.

IGF1-Stimulated Posttraumatic Hippocampal Remodeling Is Not Dependent on mTOR.

Adult hippocampal neurogenesis is stimulated acutely following traumatic brain injury (TBI). However, many hippocampal neurons born after injury develop abnormally and the number that survive long-term is debated. In experimental TBI, insulin-like growth factor-1 (IGF1) promotes hippocampal neuronal differentiation, improves immature neuron dendritic arbor morphology, increases long-term survival of neurons born after TBI, and improves cognitive function. One potential downstream mediator of the neurogenic effects of IGF1 is mammalian target of rapamycin (mTOR), which regulates proliferation as well as axonal and dendritic growth in the CNS. Excessive mTOR activation is posited to contribute to aberrant plasticity related to posttraumatic epilepsy, spurring preclinical studies of mTOR inhibitors as therapeutics for TBI. The degree to which pro-neurogenic effects of IGF1 depend upon upregulation of mTOR activity is currently unknown. Using immunostaining for phosphorylated ribosomal protein S6, a commonly used surrogate for mTOR activation, we show that controlled cortical impact TBI triggers mTOR activation in the dentate gyrus in a time-, region-, and injury severity-dependent manner. Posttraumatic mTOR activation in the granule cell layer (GCL) and dentate hilus was amplified in mice with conditional overexpression of IGF1. In contrast, delayed astrocytic activation of mTOR signaling within the dentate gyrus molecular layer, closely associated with proliferation, was not affected by IGF1 overexpression. To determine whether mTOR activation is necessary for IGF1-mediated stimulation of posttraumatic hippocampal neurogenesis, wildtype and IGF1 transgenic mice received the mTOR inhibitor rapamycin daily beginning at 3 days after TBI, following pulse labeling with bromodeoxyuridine. Compared to wildtype mice, IGF1 overexpressing mice exhibited increased posttraumatic neurogenesis, with a higher density of posttrauma-born GCL neurons at 10 days after injury. Inhibition of mTOR did not abrogate IGF1-stimulated enhancement of posttraumatic neurogenesis. Rather, rapamycin treatment in IGF1 transgenic mice, but not in WT mice, increased numbers of cells labeled with BrdU at 3 days after injury that survived to 10 days, and enhanced the proportion of posttrauma-born cells that differentiated into neurons. Because beneficial effects of IGF1 on hippocampal neurogenesis were maintained or even enhanced with delayed inhibition of mTOR, combination therapy approaches may hold promise for TBI.

Deficiency of intellectual disability-related gene Brpf1 reduced inhibitory neurotransmission in MGE-derived GABAergic interneurons

Intellectual disability is closely related to impaired GABA neurotransmission. Brpf1 was specifically expressed in medial ganglionic eminence (MGE), a developmental niche of GABAergic interneurons, and patients with BRPF1 mutations showed intellectual disability. To test its role in the development and function of MGE-derived GABAergic interneurons, we performed immunofluorescence staining, whole-cell patch-clamp, MGE transplantation, and mRNA-Seq to understand its effect on neuronal differentiation, dendritic morphology, electrophysiology, migration, and gene regulation, using mouse MGE-derived GABAergic interneurons infected with AAV-shBrpf1. The results showed that Brpf1 knockdown had a decreasing trend, although not significant, on the differentiation of GABAergic interneurons into parvalbumin+ interneurons. Moreover, increased firing threshold, decreased number of evoked action potentials, and a reduced amplitude of miniature inhibitory postsynaptic currents were observed before any significant change of MAP2+ dendritic morphology and in vivo migration ability appeared. Finally, mRNA-Seq analysis revealed that genes related to neurodevelopment and synaptic transmission such as Map2k7 were dysregulated. Our results demonstrated a key role of Brpf1 in inhibitory neurotransmission and related gene expression of GABAergic interneurons.

Amyotrophic lateral sclerosis-associated Vap33 is required for maintaining neuronal dendrite morphology and organelle distribution in Drosophila.

VAMP-associated protein (VAP) is an endoplasmic reticulum (ER) membrane protein that functions as a tethering protein at the membrane contact sites between the ER and various intracellular organelles. Mutations such as P56S in human VAPB cause neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, VAP functions in neurons are poorly understood. Here, we utilized Drosophila olfactory projection neurons with a mosaic analysis with a repressible cell marker (MARCM) to analyze the neuronal function of Vap33, a Drosophila ortholog of human VAPB. In vap33 null mutant clones, the dendrites of projection neurons exhibited defects in the maintenance of their morphology. The subcellular localization of the Golgi apparatus and mitochondria were also abnormal. These results indicate that Vap33 is required for neuronal morphology and organelle distribution. Additionally, to examine the impact of ALS-associated mutations in neurons, we overexpressed human VAPB-P56S in vap33 null mutant clones (mosaic rescue experiments) and found that, in aged flies, human VAPB-P56S expression caused mislocalization of Bruchpilot, a presynaptic protein. These results implied that synaptic protein localization and ER quality control may be affected by disease mutations. We provide insights into the physiological and pathological functions of VAP in neurons.

The potassium channel Kv4.2 regulates dendritic spine morphology, electroencephalographic characteristics and seizure susceptibility in mice

The voltage-gated potassium channel Kv4.2 is a critical regulator of dendritic excitability in the hippocampus and is crucial for dendritic signal integration. Kv4.2 mRNA and protein expression as well as function are reduced in several genetic and pharmacologically induced rodent models of epilepsy and autism. It is not known, however, whether reduced Kv4.2 is just an epiphenomenon or a disease-contributing cause of neuronal hyperexcitability and behavioral impairments in these neurological disorders. To address this question, we used male and female mice heterozygous for a Kv.2 deletion and adult-onset manipulation of hippocampal Kv4.2 expression in male mice to assess the role of Kv4.2 in regulating neuronal network excitability, morphology and anxiety-related behaviors. We observed a reduction in dendritic spine density and reduced proportions of thin and stubby spines but no changes in anxiety, overall activity, or retention of conditioned freezing memory in Kv4.2 heterozygous mice compared with wildtype littermates. Using EEG analyses, we showed elevated theta power and increased spike frequency in Kv4.2 heterozygous mice under basal conditions. In addition, the latency to onset of kainic acid-induced seizures was significantly shortened in Kv4.2 heterozygous mice compared with wildtype littermates, which was accompanied by a significant increase in theta power. By contrast, overexpressing Kv4.2 in wildtype mice through intrahippocampal injection of Kv4.2-expressing lentivirus delayed seizure onset and reduced EEG power. These results suggest that Kv4.2 is an important regulator of neuronal network excitability and dendritic spine morphology, but not anxiety-related behaviors. In the future, manipulation of Kv4.2 expression could be used to alter seizure susceptibility in epilepsy.

The spectrin-based membrane skeleton is asymmetric and remodels during neural development in C. elegans.

Perturbation of spectrin-based membrane mechanics causes hereditary elliptocytosis and spinocerebellar ataxia, but the underlying cellular basis of pathogenesis remains unclear. Here, we introduced conserved disease-associated spectrin mutations into the Caenorhabditis elegans genome and studied the contribution of spectrin to neuronal migration and dendrite formation in developing larvae. The loss of spectrin resulted in ectopic actin polymerization outside of the existing front and secondary membrane protrusions, leading to defective neuronal positioning and dendrite morphology in adult animals. Spectrin accumulated in the lateral region and rear of migrating neuroblasts and redistributes from the soma into the newly formed dendrites, indicating that the spectrin-based membrane skeleton is asymmetric and remodels to regulate actin assembly and cell shape during development. We affinity-purified spectrin from C. elegans and showed that its binding partner ankyrin functions with spectrin. Asymmetry and remodeling of the membrane skeleton might enable spatiotemporal modulation of membrane mechanics for distinct developmental events.

Primary Ciliary Deficits in the Dentate Gyrus of Fragile X Syndrome.

SummaryThe primary cilium is the non-motile cilium present in most mammalian cell types and functions as an antenna for cells to sense signals. Ablating primary cilia in postnatal newborn neurons of the dentate gyrus (DG) results in both reduced dendritic arborization and synaptic strength, leading to hippocampal-dependent learning and memory deficits. Fragile X syndrome (FXS) is a common form of inheritance for intellectual disabilities with a high risk for autism spectrum disorders, and Fmr1 KO mice, a mouse model for FXS, demonstrate deficits in newborn neuron differentiation, dendritic morphology, and memory formation in the DG. Here, we found that the number of primary cilia in Fmr1 KO mice is reduced, specifically in the DG of the hippocampus. Moreover, this cilia loss was observed postnatally mainly in newborn neurons generated from the DG, implicating that these primary ciliary deficits may possibly contribute to the pathophysiology of FXS.

TDP-43 dysfunction restricts dendritic complexity by inhibiting CREB activation and altering gene expression

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative diseases that present with similar TDP-43 pathology in patient tissue. TDP-43 is an RNA-binding protein which forms aggregates in neurons of ALS and FTD patients as well as in a subset of patients diagnosed with other neurodegenerative diseases. Despite our understanding that TDP-43 is essential for many aspects of RNA metabolism, it remains obscure how TDP-43 dysfunction contributes to neurodegeneration. Interestingly, altered neuronal dendritic morphology is a common theme among several neurological disorders and is thought to precede neurodegeneration. We previously found that both TDP-43 overexpression (OE) and knockdown (KD) result in reduced dendritic branching of cortical neurons. In this study, we used TRIBE (targets of RNA-binding proteins identified by editing) as an approach to identify signaling pathways that regulate dendritic branching downstream of TDP-43. We found that TDP-43 RNA targets are enriched for pathways that signal to the CREB transcription factor. We further found that TDP-43 dysfunction inhibits CREB activation and CREB transcriptional output, and restoring CREB signaling rescues defects in dendritic branching. Finally, we demonstrate, using RNA sequencing, that TDP-43 OE and KD cause similar changes in the abundance of specific messenger RNAs, consistent with their ability to produce similar morphological defects. Our data therefore provide a mechanism by which TDP-43 dysfunction interferes with dendritic branching, and may define pathways for therapeutic intervention in neurodegenerative diseases.

CD40L Reverse Signaling Influences Dendrite Spine Morphology and Expression of PSD-95 and Rho Small GTPases.

CD40-activated CD40L reverse signaling is a major physiological regulator of neural process growth from many kinds of developing neurons. Here we have investigated whether CD40L-reverse signaling also influences dendrite spine number and morphology in striatal medium spiny neurons (MSNs). Golgi preparations revealed no differences in the spine density, but because the dendrite arbors of MSNs were larger and branched in Cd40–/– mice, the total number of spines was greater in Cd40–/– mice. We also detected more mature spines compared with wild-type littermates. Western blot revealed that MSN cultures from Cd40–/– mice had significantly less PSD-95 and there were changes in RhoA/B/C and Cdc42. Immunocytochemistry revealed that PSD-95 was clustered in spines in Cd40–/– neurons compared with more diffuse labeling in Cd40+/+ neurons. Activation of CD40L-reverse signaling with CD40-Fc prevented the changes observed in Cd40–/– cultures. Our findings suggest that CD40L-reverse signaling influences dendrite spine morphology and related protein expression and distribution.

3D mesh processing using GAMer 2 to enable reaction-diffusion simulations in realistic cellular geometries.

Recent advances in electron microscopy have enabled the imaging of single cells in 3D at nanometer length scale resolutions. An uncharted frontier for in silico biology is the ability to simulate cellular processes using these observed geometries. Enabling such simulations requires watertight meshing of electron micrograph images into 3D volume meshes, which can then form the basis of computer simulations of such processes using numerical techniques such as the finite element method. In this paper, we describe the use of our recently rewritten mesh processing software, GAMer 2, to bridge the gap between poorly conditioned meshes generated from segmented micrographs and boundary marked tetrahedral meshes which are compatible with simulation. We demonstrate the application of a workflow using GAMer 2 to a series of electron micrographs of neuronal dendrite morphology explored at three different length scales and show that the resulting meshes are suitable for finite element simulations. This work is an important step towards making physical simulations of biological processes in realistic geometries routine. Innovations in algorithms to reconstruct and simulate cellular length scale phenomena based on emerging structural data will enable realistic physical models and advance discovery at the interface of geometry and cellular processes. We posit that a new frontier at the intersection of computational technologies and single cell biology is now open.

Protein phosphatase PP2A regulates microtubule orientation and dendrite pruning in Drosophila.

Pruning that selectively eliminates inappropriate projections is crucial for sculpting neural circuits during development. During Drosophila metamorphosis, ddaC sensory neurons undergo dendrite-specific pruning in response to the steroid hormone ecdysone. However, the understanding of the molecular mechanisms underlying dendrite pruning remains incomplete. Here, we show that protein phosphatase 2A (PP2A) is required for dendrite pruning. The catalytic (Microtubule star/Mts), scaffolding (PP2A-29B), and two regulatory subunits (Widerborst/Wdb and Twins/Tws) play important roles in dendrite pruning. Functional analyses indicate that PP2A, via Wdb, facilitates the expression of Sox14 and Mical prior to dendrite pruning. Furthermore, PP2A, via Tws, governs the minus-end-out orientation of microtubules (MTs) in the dendrites. Moreover, the levels of Klp10A, a MT depolymerase, increase when PP2A is compromised. Attenuation of Klp10A fully rescues the MT orientation defects in mts or pp2a-29b RNAi ddaC neurons, suggesting that PP2A governs dendritic MT orientation by suppressing Klp10A levels and/or function. Taken together, this study sheds light on a novel function of PP2A in regulating dendrite pruning and dendritic MT polarity in sensory neurons.