Dendrites Research Articles

GM‐CSF Treatment Increases the Number of Purkinje Cells in a Mouse Model of Down Syndrome

AbstractBackgroundDown syndrome (DS), the most common chromosomal disorder that is a genetic cause of intellectual disability (ID), is characterized by mild‐to‐severe cognitive deficits and several neurological and brain anatomical abnormalities in people with DS and in animal models of DS. Among them, a marked reduction in the volume of the cerebellum has been reported in fetuses, newborns, and adults with DS, and in mouse models of DS. It has been well documented that children and adults with DS display motor deficits consistent with cerebellar abnormalities. Growing evidence suggests that the cerebellum is involved in the processing of cognitive functions. Reduced size of the cerebellum may be one of the underlying causes of cognitive deficits in DS. However, the cellular functions of the cerebellum remain poorly understood in people with DS. The beneficial effects of treatment with the hematopoietic growth factor granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in animal models of DS, Alzheimer’s disease (AD), normal aging, and clinical trials of AD subjects suggest that it may serve as a potential therapeutic for these disorders. Our recent published data showed that treatment with GM‐CSF rescues the cognitive impairments and improves hippocampal pathology in Dp16 mice. To elucidate cerebellum‐associated cellular functions, we investigated purkinje cell pathology in aged male Dp16 mice after treatment with GM‐CSF compared to saline‐injected mice.MethodWe injected 12‐ to 14‐month‐old male Dp16 male mice subcutaneously with GM‐CSF (5 mg/day; 5 days/week) or with saline (200 ml) for 24 injections total over 29 days. On day 29, the brains were fixed, and tissues were processed for immunohistochemistry.ResultWe observed a reduced number of parvalbumin‐positive purkinje cells and loss of arborization in the cerebellum of male Dp16 mice compared to wild‐type littermates. Treatment with GM‐CSF significantly increased the number of parvalbumin‐positive purkinje cells and partially restored dendritic arborization in the cerebellum of Dp16 mice compared to saline‐injected mice.ConclusionOur results suggest that treatment with GM‐CSF improves cellular abnormalities by increasing the number of purkinje cells in the cerebellum of Dp16 mice. Taken together with our previous results, these findings provide further evidence that GM‐CSF/sargramostim may have therapeutic benefits in people with DS.

Regulation of extracellular progranulin in the medial prefrontal cortex

AbstractBackgroundLoss‐of‐function mutations in progranulin (GRN) are a major genetic cause of frontotemporal dementia (FTD). Progranulin is a secreted protein that is trafficked to lysosomes, where it promotes lysosomal function. Progranulin also exerts neurotrophic and anti‐inflammatory effects, some of which may be mediated by extracellular signaling. Neurons and microglia express progranulin and loss of progranulin from either cell type induces FTD‐like deficits in mice. Both cell types secrete progranulin in culture, but their contribution to extracellular progranulin in the brain is unclear, as are the mechanisms regulating progranulin secretion from each cell type. In this study, we used microdialysis to gain insight into the contribution of neurons and microglia to extracellular progranulin in the medial prefrontal cortex (mPFC), a region that degenerates in FTD and develops impaired dendritic arborization in progranulin‐insufficient mice.MethodPolyethylene probes (2 mm, 1000 kDa cut‐off) were implanted in mouse mPFC and perfused with artificial cerebrospinal fluid containing 4% BSA using a push‐pull pump system (Atmos LM, Amuza). Progranulin levels in interstitial fluid (ISF) were measured by ELISA (Adipogen).ResultGrn+/– mice exhibited a roughly 50% decrease in ISF progranulin versus wild‐type, which was similar to the reduction of progranulin in mPFC tissue. In wild‐type mice, inducing cellular depolarization with KCl (100 mM) increased ISF progranulin, but stimulating synaptic activity with picrotoxin (100 μM) or NMDA (50 μM) did not. Inducing systemic inflammation with LPS (10 mg/kg i.p.) increased ISF progranulin to around 200% of baseline at eight hours after injection. Analysis of ISF progranulin from microglial progranulin knockout mice (Cx3Cr1‐Cre‐ER:Grnfl/fl) revealed a nearly 50% reduction in ISF progranulin compared to Cre– littermates, which was a larger reduction than observed in mPFC tissue. Ongoing studies are analyzing ISF progranulin in neuronal progranulin knockout mice (CamKII‐Cre:Grnfl/fl).ConclusionOur findings indicate that inflammation increases ISF progranulin in the mPFC and that nearly half of the extracellular progranulin in the mPFC may originate from microglia. In contrast, synaptic activity does not significantly increase ISF progranulin in the mPFC. Ongoing studies will investigate the contribution of neurons to extracellular progranulin in the mPFC.

Reduced expression of GluN2A induces a delay in neuron maturation.

NMDA receptors (NMDARs) play an important role in synaptic plasticity both in physiological and pathological conditions. GluN2A and GluN2B are the most expressed NMDAR regulatory subunits, in the hippocampus and other cognitive-related brain structures. GluN2B is characteristic of immature structures and GluN2A of mature ones. Changes in GluN2A expression were associated with complex phenotypes that led to complex neurodevelopmental disorders, including the occurrence of seizures. However, little is known about the role of GluN2A in these phenotypes. In this work, we reduced GluN2A expression in mature neuronal cultures and observed an altered GluN2A/GluN2B ratio. Furthermore, those neurons exhibit an increase in immature dendritic spines and dendritic branching, as well as an increased response to glutamate stimulus. This phenotype (considering GluN2A/GluN2B ratio, index branching and glutamate response) resembles those observed at immature neuronal stages invitro. We propose that this immature phenotype led to a higher response to glutamate stimulus which, invivo, would be the basis of reduced threshold for seizure onset in GluN2A-pathological conditions.

Dendritic arbor dynamics and stability in health and disease.

Dendritogenesis, a process of dendritic arbor development, is essential for the formation of functional neuronal networks, and in mammals, it begins in early life and continues into adulthood. It is a highly dynamic process in which dendritic branches form and regress until mature connectivity is achieved. Thereafter, dendritic branches are considered stable and do not undergo substantial rearrangements, although several exceptions have been described in the literature. After this long period of relative stability, significant changes in dendritic branching occur when the brain begins to age. Several neurological diseases, occurring both during development and in adulthood, have severe effects on the morphology of dendritic arbors, often associated with intellectual dysfunction. The molecular mechanisms of dendritogenesis are fairly well described. In contrast, knowledge of the molecular mechanisms of dendritic arbor stabilization and pathology‑induced instability is still quite incomplete, and several important questions remain unanswered. We describe the dynamic changes during development and adulthood and in different pathologies. Whenever possible, we also provide details on the molecular mechanisms behind dendritic dynamics and stability.

Responses to Pattern-Violating Visual Stimuli Evolve Differently Over Days in Somata and Distal Apical Dendrites.

Scientists have long conjectured that the neocortex learns patterns in sensory data to generate top-down predictions of upcoming stimuli. In line with this conjecture, different responses to pattern-matching vs pattern-violating visual stimuli have been observed in both spiking and somatic calcium imaging data. However, it remains unknown whether these pattern-violation signals are different between the distal apical dendrites, which are heavily targeted by top-down signals, and the somata, where bottom-up information is primarily integrated. Furthermore, it is unknown how responses to pattern-violating stimuli evolve over time as an animal gains more experience with them. Here, we address these unanswered questions by analyzing responses of individual somata and dendritic branches of layer 2/3 and layer 5 pyramidal neurons tracked over multiple days in primary visual cortex of awake, behaving female and male mice. We use sequences of Gabor patches with patterns in their orientations to create pattern-matching and pattern-violating stimuli, and two-photon calcium imaging to record neuronal responses. Many neurons in both layers show large differences between their responses to pattern-matching and pattern-violating stimuli. Interestingly, these responses evolve in opposite directions in the somata and distal apical dendrites, with somata becoming less sensitive to pattern-violating stimuli and distal apical dendrites more sensitive. These differences between the somata and distal apical dendrites may be important for hierarchical computation of sensory predictions and learning, since these two compartments tend to receive bottom-up and top-down information, respectively.

Vitamin B12-folic acid supplementation improves memory by altering mitochondrial dynamics, dendritic arborization, and neurodegeneration in old and amnesic male mice

Memory impairment during aging and amnesia is attributed to compromised mitochondrial dynamics and mitophagy and other mitochondrial quality control mechanisms. Mitochondrial dynamics involves the continuous process of fission and fusion of mitochondria within a cell and is a fundamental mechanism for regulating mitochondrial quality and function. An extensive range of potential nutritional supplements has been shown to improve mitochondrial health, synaptic plasticity, and cognitive functions. Previous findings revealed that supplementation of vitamin B12-folic acid reduces locomotor deficits and mitochondrial abnormalities but enhances mitochondrial and neuronal health. The present study aims to explore the impact of combined vitamin B12-folic acid supplementation on mitochondrial dynamics, neuronal health, and memory decline in old age and scopolamine-induced amnesia, which remains elusive. The results demonstrated that supplementation led to a noteworthy increase in recognition and spatial memory and expression of memory-related protein BDNF in old and amnesic mice. Moreover, the decrease in the fragmented mitochondrial number was validated by the downregulation of mitochondrial fission p-Drp1 (S616) protein and the increase in elongated mitochondria by the upregulation of mitochondrial fusion Mfn2 protein. The increased spine density and dendritic arborization in old and amnesic mice upon supplementation were confirmed by the enhanced expression level of PSD95 and synaptophysin. Furthermore, supplementation reduced ROS production, inhibited Caspase-3 activation, mitigated neurodegeneration, and enhanced mitochondrial membrane potential, ATP production, Vdac1 expression, myelination, in old and amnesic mice. Collectively, our findings imply that combined supplementation of vitamin B12-folic acid improves mitochondrial dynamics and neuronal health, and leads to recovery of memory during old age and amnesia.

Gulf War toxicant-induced reductions in dendritic arbors and spine densities of dentate granule cells are improved by treatment with a Nrf2 activator

Gulf War Illness (GWI) is a chronic multi-symptom disorder affecting approximately 30 % of Veterans deployed to the Persian Gulf from 1990 to 91. GWI encompasses a wide spectrum of symptoms which frequently include neurological problems such as learning and memory impairments, mood disorders, and an increased incidence of neurodegenerative disorders. Combined exposure to both reversible and irreversible acetylcholinesterase (AChE) inhibitors has been identified as a likely risk factor for GWI. It is possible that the exposures affected connectivity in the brain, and it was also unknown whether this could benefit from treatment. We assessed chronic changes in dendritic architecture in granule cells of the dentate gyrus following exposure to pyridostigmine bromide (PB, 0.7 mg/kg), chlorpyrifos (CPF, 12.5 mg/kg), and N,N-diethyl-m-toluamide (DEET, 7.5 mg/kg) in male C57Bl/6J mice. We also evaluated the therapeutic effects of dietary administration for eight weeks of 1 % tert-butylhydroquinone (tBHQ), a Nrf2 activator, on long-term neuronal morphology. We found that Gulf War toxicant exposure resulted in reduced dendritic length and branching as well as overall spine density in dentate granule cells at 14 weeks post-exposure and that these effects were ameliorated by treatment with tBHQ. These findings indicate that Gulf War toxicant exposure results in chronic changes to dentate granule cell morphology and that modulation of neuroprotective transcription factors such as Nrf2 may improve long-term neuronal health in the hippocampus.

Neuronal dysfunction caused by FUSR521G promotes ALS-associated phenotypes that are attenuated by NF-κB inhibition

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are related neurodegenerative diseases that belong to a common disease spectrum based on overlapping clinical, pathological and genetic evidence. Early pathological changes to the morphology and synapses of affected neuron populations in ALS/FTD suggest a common underlying mechanism of disease that requires further investigation. Fused in sarcoma (FUS) is a DNA/RNA-binding protein with known genetic and pathological links to ALS/FTD. Expression of ALS-linked FUS mutants in mice causes cognitive and motor defects, which correlate with loss of motor neuron dendritic branching and synapses, in addition to other pathological features of ALS/FTD. The role of ALS-linked FUS mutants in causing ALS/FTD-associated disease phenotypes is well established, but there are significant gaps in our understanding of the cell-autonomous role of FUS in promoting structural changes to motor neurons, and how these changes relate to disease progression. Here we generated a neuron-specific FUS-transgenic mouse model expressing the ALS-linked human FUSR521G variant, hFUSR521G/Syn1, to investigate the cell-autonomous role of FUSR521G in causing loss of dendritic branching and synapses of motor neurons, and to understand how these changes relate to ALS-associated phenotypes. Longitudinal analysis of mice revealed that cognitive impairments in juvenile hFUSR521G/Syn1 mice coincide with reduced dendritic branching of cortical motor neurons in the absence of motor impairments or changes in the neuromorphology of spinal motor neurons. Motor impairments and dendritic attrition of spinal motor neurons developed later in aged hFUSR521G/Syn1 mice, along with FUS cytoplasmic mislocalisation, mitochondrial abnormalities and glial activation. Neuroinflammation promotes neuronal dysfunction and drives disease progression in ALS/FTD. The therapeutic effects of inhibiting the pro-inflammatory nuclear factor kappa B (NF-κB) pathway with an analog of Withaferin A, IMS-088, were assessed in symptomatic hFUSR521G/Syn1 mice and were found to improve cognitive and motor function, increase dendritic branches and synapses of motor neurons, and attenuate other ALS/FTD-associated pathological features. Treatment of primary cortical neurons expressing FUSR521G with IMS-088 promoted the restoration of dendritic mitochondrial numbers and mitochondrial activity to wild-type levels, suggesting that inhibition of NF-κB permits the restoration of mitochondrial stasis in our models. Collectively, this work demonstrates that FUSR521G has a cell-autonomous role in causing early pathological changes to dendritic and synaptic structures of motor neurons, and that these changes precede motor defects and other well-known pathological features of ALS/FTD. Finally, these findings provide further support that modulation of the NF-κB pathway in ALS/FTD is an important therapeutic approach to attenuate disease.

S-SCAM is essential for synapse formation.

Synapse formation is critical for the wiring of neural circuits in the developing brain. The synaptic scaffolding protein S-SCAM/MAGI-2 has important roles in the assembly of signaling complexes at post-synaptic densities. However, the role of S-SCAM in establishing the entire synapse is not known. Here, we report significant effects of RNAi-induced S-SCAM knockdown on the number of synapses in early stages of network development in vitro. In vivo knockdown during the first three postnatal weeks reduced the number of dendritic spines in the rat brain neocortex. Knockdown of S-SCAM in cultured hippocampal neurons severely reduced the clustering of both pre- and post-synaptic components. This included synaptic vesicle proteins, pre- and post-synaptic scaffolding proteins, and cell adhesion molecules, suggesting that entire synapses fail to form. Correspondingly, functional and morphological characteristics of developing neurons were affected by reducing S-SCAM protein levels; neurons displayed severely impaired synaptic transmission and reduced dendritic arborization. A next-generation sequencing approach showed normal expression of housekeeping genes but changes in expression levels in 39 synaptic signaling molecules in cultured neurons. These results indicate that S-SCAM mediates the recruitment of all key classes of synaptic molecules during synapse assembly and is critical for the development of neural circuits in the developing brain.

Genomic glucocorticoid receptor effects guide acute stress-induced delayed anxiety and basolateral amygdala spine plasticity in rats

Anxiety, a state related to anticipatory fear, can be adaptive in the face of environmental threats or stressors. However, anxiety can also become persistent and manifest as anxiety- and stress-related disorders, such as generalized anxiety or post-traumatic stress disorder (PTSD). In rodents, systemic administration of glucocorticoids (GCs) or short-term restraint stress induces anxiety-like behaviors and dendritic branching within the basolateral complex of the amygdala (BLA) ten days later. Additionally, increased arousal-related memory retention mediated by elevated GCs requires concomitant noradrenaline (NE) signaling, both acting in the BLA. It is unknown whether GCs and NE play a role in the delayed acute stress-induced effects on behavior and BLA dendritic plasticity. Here, inhibiting corticosterone (CORT) elevation during 2 h of restraint stress prevents stress-induced increases in delayed anxiety-like behavior and BLA dendritic spine density in rats. Also, we show that the delayed acute stress-induced effects on behavior and morphological alterations are critically dependent on genomic glucocorticoid receptor (GR) actions in the BLA. Unlike CORT, the pharmacological enhancement of NE signaling in the BLA was insufficient to drive delayed anxiety-related behavior. Nonetheless, the delayed anxiety-like behavior ten days after acute stress requires NE signaling in the BLA during stress exposure. Therefore, we define the essential roles of two stress-related hormones for the late stress consequences, acting at two separate times: CORT, via GR, immediately during stress, and NE, via beta-adrenoceptors, during the expression of delayed anxiety.

Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons.

Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. We investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies. We used CRISPR-Cas9 genome engineering approaches to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Fluorescently tagged endogenous Ppk1 localizes to dendrites, as previously reported, and, unexpectedly, to axons and axon terminals. In dendrites, Ppk1 is present throughout actively growing dendrite branches and is stably integrated into the neuronal cell membrane during the expansive growth of the arbor. Although Ppk channels are dispensable for dendrite growth, we found that an over-active channel mutant severely reduces dendrite growth, likely by acting at an internal membrane and not the dendritic membrane. Our data reveal that the molecular motor dynein and recycling endosome GTPase Rab11 are needed for the proper trafficking of Ppk1 to dendrites. Based on our data, we propose that Ppk channel transport is coordinated with dendrite morphogenesis, which ensures proper ion channel density and distribution in sensory dendrites.

Prenatal folic acid and vitamin B12 imbalance alter neuronal morphology and synaptic density in the mouse neocortex

Previous reports have provided evidence that insufficient or excessive maternal folic acid (FA) intake during pregnancy can alter neurodevelopment of the offspring by modulating prenatal neurogenesis. Furthermore, our earlier work in a mouse model confirmed long-term structural changes at the cellular level of either deficient or excessive FA supply by comparably reducing dendritic arborization of cortical projection neurons. Here, we report that excessive amounts of FA decrease arborization of deep layer projection neurons, but not upper layer neurons and that reduced complexity of deep layer neurons is not observed when folic acid is replaced by folinic acid, a stable reduced form of folate. In addition, deficiency of B12, a vitamin that critically regulates folate metabolism, causes even more marked decreases in neuronal arborization in both deep and upper layer neurons and particularly in combination with FA excess. Furthermore, both FA excess and B12 deficiency affect synaptic density and morphology. Our findings point to neurodevelopmental risks associated with insufficient amounts of prenatal B12, particularly in association with high levels of FA intake, suggesting that the neurodevelopmental program is sensitive to an imbalance in the status of these interacting micronutrients.

Ventral posterolateral and ventral posteromedial thalamocortical neurons have distinct physiological properties.

Somatosensory information is propagated from the periphery to the cerebral cortex by two parallel pathways through the ventral posterolateral (VPL) and ventral posteromedial (VPM) thalamus. VPL and VPM neurons receive somatosensory signals from the body and head, respectively. VPL and VPM neurons may also receive cell type-specific GABAergic input from the reticular nucleus of the thalamus. Although VPL and VPM neurons have distinct connectivity and physiological roles, differences in their functional properties remain unclear as they are often studied as one ventrobasal thalamus neuron population. Here, we directly compared synaptic and intrinsic properties of VPL and VPM neurons in C57Bl/6J mice of both sexes aged P25-P32. VPL neurons showed greater depolarization-induced spike firing and spike frequency adaptation than VPM neurons. VPL and VPM neurons fired similar numbers of spikes during hyperpolarization rebound bursts, but VPM neurons exhibited shorter burst latency compared with VPL neurons, which correlated with larger sag potential. VPM neurons had larger membrane capacitance and more complex dendritic arbors. Recordings of spontaneous and evoked synaptic transmission suggested that VPL neurons receive stronger excitatory synaptic input, whereas inhibitory synapse strength was stronger in VPM neurons. This work indicates that VPL and VPM thalamocortical neurons have distinct intrinsic and synaptic properties. The observed functional differences could have important implications for their specific physiological and pathophysiological roles within the somatosensory thalamocortical network.NEW & NOTEWORTHY This study revealed that somatosensory thalamocortical neurons in the VPL and VPM have substantial differences in excitatory synaptic input and intrinsic firing properties. The distinct properties suggest that VPL and VPM neurons could process somatosensory information differently and have selective vulnerability to disease. This work improves our understanding of nucleus-specific neuron function in the thalamus and demonstrates the critical importance of studying these parallel somatosensory pathways separately.

Sex-specific dendritic morphology of hippocampal pyramidal neurons in the adolescent and young adult rats.

CA1 and CA3 pyramidal neurons are the major sources of hippocampal efferents. The structural features of these neurons are presumed to be involved in various normal/abnormal cognitive and emotional outcomes by influencing the pattern of synaptic inputs and neuronal signal processing. Although many studies have described hippocampal structure differences between males and females, these reports mainly focused on gross anatomical features in adult or aged models, and such distinctions on neuronal morphology and dendritic spine density during adolescence, a period of high vulnerability to neurodevelopmental disorders, have received much less attention. In this work, we analyzed dendritic architecture and density of spines in CA1 and CA3 neurons of male and female rats in early adolescence (postnatal day, PND 40) and compared them with those in late adolescence/young adulthood (PND 60). On PND 40, CA1 neurons of male rats showed more Sholl intersections and spine density in apical and basal dendrites compared to those in females. The Sholl intersections in basal dendrites of CA3 neurons were also more in males, whereas the number of apical dendrite intersections was not significantly different between sexes. In male rats, there was a notable decrease in the number of branch and terminal points in the basal dendrite of CA1 neurons of young adults when compared to their sex-matched adolescent rats. On the other hand, CA1 neurons in young adult females also showed more Sholl intersections in apical and basal dendrites compared to adolescent females. Meanwhile, the total cable length, the number of branches, and terminal points of apical dendrites in CA3 neurons also exhibited a significant reduction in young adult male rats compared to their sex-matched adolescents. In young adult rats, both apical and basal dendrites of CA3 neurons in males showed fewer intersections with Sholl circles, but there were no significant differences in dendritic spine density or count estimation between males and females. On the other hand, young adult female rats had more Sholl intersections and dendritic spine count on the basal dendrites of CA3 neurons compared to adolescent females. Although no significant sex- and age-dependent difference in neuronal density was detected in CA1 and CA3 subareas, CA3 pyramidal neurons of both male and female rats showed reduced soma area compared to adolescent rats. Our findings show that the sex differences in the dendritic structure of CA1 and CA3 neurons vary by age and also by the compartments of dendritic arbors. Such variations in the morphology of hippocampal pyramidal neurons may take part as a basis for normal cognitive and affective differences between the sexes, as well as distinct sensitivity to interfering factors and the prevalence of neuropsychological diseases.

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.

Structural-functional properties of direct-pathway striatal neurons at early and chronic stages of dopamine denervation.

The dendritic arbour of striatal projection neurons (SPNs) is the primary anatomical site where dopamine and glutamate inputs to the basal ganglia functionally interact to control movement. These dendritic arbourisations undergo atrophic changes in Parkinson's disease. A reduction in the dendritic complexity of SPNs is found also in animal models with severe striatal dopamine denervation. Using 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle as a model, we set out to compare morphological and electrophysiological properties of SPNs at an early versus a chronic stage of dopaminergic degeneration. Ex vivo recordings were performed in transgenic mice where SPNs forming the direct pathway (dSPNs) express a fluorescent reporter protein. At both the time points studied (5 and 28 days following 6-OHDA lesion), there was a complete loss of dopaminergic fibres through the dorsolateral striatum. A reduction in dSPN dendritic complexity and spine density was manifest at 28, but not 5days post-lesion. At the late time point, dSPN also exhibited a marked increase in intrinsic excitability (reduced rheobase current, increased input resistance, more evoked action potentials in response to depolarising currents), which was not present at 5days. The increase in neuronal excitability was accompanied by a marked reduction in inward-rectifying potassium (Kir) currents (which dampen the SPN response to depolarising stimuli). Our results show that dSPNs undergo delayed coordinate changes in dendritic morphology, intrinsic excitability and Kir conductance following dopamine denervation. These changes are predicted to interfere with the dSPN capacity to produce a normal movement-related output.

Microstructure and properties of the GEWZ522K casting magnesium alloy based on the Mg–Gd–Nd–Y–Zn–Zr system

The article discusses the solidification and phase composition of the (wt.%) Mg–4.8Gd–2.1Nd–1.6Y–0.4Zn–0.6Zr (GEWZ522K) casting alloy. It is demonstrated that in the as-cast state, the alloy structure comprises primary zirconium particles, dendrites of the magnesium solid solution (αMg), and eutectic intermetallic phases located between dendritic branches. Following solution heat treatment at t = 530±5 °C, the alloy transitions into a single-phase state and can be significantly strengthened through artificial aging after quenching. It is recommended to apply alloy aging at t = 250 °C for 8–10 h or at t = 200 °C for 15–18 h. This approach leads to the maximum strengthening of the alloy, with the best mechanical properties achieved for the alloy aged at t = 250 °C. Regardless of the aging method used, the ultimate tensile strength (UTS) of the samples surpasses 300 MPa, which significantly exceeds that of commercial casting alloys according to GOST 2856-79. The measured corrosion rate for the GEWZ522K alloy is 7.5±0.4 mm/year, that slightly higher than that for the less alloyed commercial alloy ML10 (approximately 2.5 mm/year) tested under similar conditions. Furthermore, the alloy was subjected to tests for ignition resistance when in contact with air. It was observed that with continuous airflow over the specimen’s surface, ignition centers appear at t = 625 °C due to the breakdown of the oxide film, causing the alloy to nearly completely melt. Therefore, the GEWZ522K alloy can be employed as a high-strength casting alloy. However, during the operation of cast parts, particular attention must be paid to safeguarding the surface of these parts against corrosion.

Panoramic variation analysis of a family with neurodevelopmental disorders caused by biallelic loss-of-function variants in TMEM141, DDHD2, and LHFPL5.

Highly clinical and genetic heterogeneity of neurodevelopmental disorders presents a major challenge in clinical genetics and medicine. Panoramic variation analysis is imperative to analyze the disease phenotypes resulting from multilocus genomic variation. Here, a Pakistani family with parental consanguinity was presented, characterized with severe intellectual disability (ID), spastic paraplegia, and deafness. Homozygosity mapping, integrated single nucleotide polymorphism (SNP) array, whole-exome sequencing, and whole-genome sequencing were performed, and homozygous variants in TMEM141 (c.270G>A, p.Trp90*), DDHD2 (c.411+767_c.1249-327del), and LHFPL5 (c.250delC, p.Leu84*) were identified. A Tmem141p.Trp90*/p.Trp90* mouse model was generated. Behavioral studies showed impairments in learning ability and motor coordination. Brain slice electrophysiology and Golgi staining demonstrated deficient synaptic plasticity in hippocampal neurons and abnormal dendritic branching in cerebellar Purkinje cells. Transmission electron microscopy showed abnormal mitochondrial morphology. Furthermore, studies on a human in vitro neuronal model (SH-SY5Y cells) with stable shRNA-mediated knockdown of TMEM141 showed deleterious effect on bioenergetic function, possibly explaining the pathogenesis of replicated phenotypes in the cross-species mouse model. Conclusively, panoramic variation analysis revealed that multilocus genomic variations of TMEM141, DDHD2, and LHFPL5 together caused variable phenotypes in patient. Notably, the biallelic loss-of-function variants of TMEM141 were responsible for syndromic ID.

The Role of Protocadherin γ in Adult Sensory Neurons and Skin Reinnervation.

The clustered protocadherins (cPcdhs) play a critical role in the patterning of several CNS axon and dendritic arbors, through regulation of homophilic self and neighboring interactions. While not explored, primary peripheral sensory afferents that innervate the epidermis may require similar constraints to convey spatial signals with appropriate fidelity. Here, we show that members of the γ-Pcdh (Pcdhγ) family are expressed in both adult sensory neuron axons and in neighboring keratinocytes that have close interactions during skin reinnervation. Adult mice of both sexes were studied. Pcdhγ knock-down either through small interfering RNA (siRNA) transduction or AAV-Cre recombinase transfection of adult mouse primary sensory neurons from floxed Pcdhγ mice was associated with a remarkable rise in neurite outgrowth and branching. Rises in outgrowth were abrogated by Rac1 inhibition. Moreover, AAV-Cre knock-down in Pcdhγ floxed neurons generated a rise in neurite self-intersections, and a robust rise in neighbor intersections or tiling, suggesting a role in sensory axon repulsion. Interestingly, preconditioned (3-d axotomy) neurons with enhanced growth had temporary declines in Pcdhγ and lessened outgrowth from Pcdhγ siRNA. In vivo, mice with local hindpaw skin Pcdhγ knock-down by siRNA had accelerated reinnervation by new epidermal axons with greater terminal branching and reduced intra-axonal spacing. Pcdhγ knock-down also had reciprocal impacts on keratinocyte density and nuclear size. Taken together, this work provides evidence for a role of Pcdhγ in attenuating outgrowth of sensory axons and their interactions, with implications in how new reinnervating axons following injury fare amid skin keratinocytes that also express Pcdhγ.SIGNIFICANCE STATEMENT The molecular mechanisms and potential constraints that govern skin reinnervation and patterning by sensory axons are largely unexplored. Here, we show that γ-protocadherins (Pcdhγ) may help to dictate interaction not only among axons but also between axons and keratinocytes as the former re-enter the skin during reinnervation. Pcdhγ neuronal knock-down enhances outgrowth in peripheral sensory neurons, involving the growth cone protein Rac1 whereas skin Pcdhγ knock-down generates rises in terminal epidermal axon growth and branching during re-innervation. Manipulation of sensory axon regrowth within the epidermis offers an opportunity to influence regenerative outcomes following nerve injury.

Sustained OMA1-mediated integrated stress response is beneficial for spastic ataxia type 5.

AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane. Heterozygous AFG3L2 mutations cause spinocerebellar ataxia type 28 (SCA28) or dominant optic atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe spastic ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the overactivation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the inner mitochondrial membrane protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response. In general, the integrated stress response reduces global protein synthesis and drives the expression of cytoprotective genes that allow cells to endure proteotoxic stress. However, the relevance of the OMA1-DELE1-HRI axis in vivo, and especially in a human CNS disease context, has been poorly documented thus far. In this work, we demonstrated that mitochondrial proteotoxicity in the absence/mutation of AFG3L2 activates the OMA1-DELE1-HRI pathway eliciting the integrated stress response. We found enhanced OMA1-dependent processing of DELE1 upon depletion of AFG3L2. Also, in both skin fibroblasts from SPAX5 patients (including a novel case) and in the cerebellum of Afg3l2-/- mice we detected increased phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), increased levels of ATF4 and strong upregulation of its downstream targets (Chop, Chac1, Ppp1r15a and Ffg21). Silencing of DELE1 or HRI in SPAX5 fibroblasts (where OMA1 is overactivated at basal state) reduces eIF2α phosphorylation and affects cell growth. In agreement, pharmacological potentiation of integrated stress response via Sephin-1, a drug that selectively inhibits the stress-induced eIF2alpha phosphatase GADD34 (encoded by Ppp1r15a), improved cell growth of SPAX5 fibroblasts and cell survival and dendritic arborization ex vivo in primary Afg3l2-/- Purkinje neurons. Notably, Sephin-1 treatment in vivo extended the lifespan of Afg3l2-/- mice, improved Purkinje neuron morphology, mitochondrial ultrastructure and respiratory capacity. These data indicate that activation of the OMA1-DELE1-HRI pathway is protective in the context of SPAX5. Pharmacological tuning of the integrated stress response may represent a future therapeutic strategy for SPAX5 and other cerebellar ataxias caused by impaired mitochondrial proteostasis.