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Astrocyte-microglia crosstalk is vital for neuronal survival and clearing aggregate accumulation in neurodegenerative diseases. While interleukin-3 (IL-3) has been reported to exert both protective and detrimental effects in neurodegenerative diseases, however, its role in α-synuclein pathology remains unclear. In this study, it is found that astrocytic IL-3 and microglial IL-3R are positively responsive to α-synuclein pathology in the brains of transgenic A53T Parkinson's disease (PD) mice and in an adeno-associated virus (AAV)-human α-synuclein (AAV-hα-Syn)-injected PD mouse model. Exogenous IL-3 infusion reduces behavioral abnormities and nigrostriatal α-synuclein pathology. Mechanistically, IL-3 induces microglial phagocytosis of pathological α-synuclein while simultaneously stimulating dopaminergic (DA) neurons to clear pathological α-synuclein via induction of autophagy through the IFN-ß/Irgm1 pathway. Due to its limited efficiency in crossing the blood-brain barrier, a precise IL-3 delivery strategy is developed by cross-linking IL-3 and RVG29 with PEG-Linker (RVG-modified IL-3 nanogels-RVG-IL3 NGs). Intravenous administration of RVG-IL3 NGs shows efficient uptake by microglia and DA neurons within the brain. RVG-IL3 NGs ameliorate motor deficits and pathological α-synuclein by improving microglial and neuronal function in the AAV-hα-Syn mouse model of PD. Collectively, IL-3 may represent a feasible therapeutic strategy for PD.
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While an association between Parkinson's disease (PD) and viral infections has been recognized, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on PD progression remains unclear. Here, we demonstrate that SARS-CoV-2 infection heightens the risk of PD using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons and a human angiotensin-converting enzyme 2 (hACE2) transgenic (Tg) mouse model. Our findings reveal that SARS-CoV-2 infection exacerbates PD susceptibility and cellular toxicity in DA neurons pre-treated with human preformed fibrils (hPFFs). Additionally, nasally delivered SARS-CoV-2 infects DA neurons in hACE2 Tg mice, aggravating the damage initiated by hPFFs. Mice infected with SARS-CoV-2 display persisting neuroinflammation even after the virus is no longer detectable in the brain. A comprehensive analysis suggests that the inflammatory response mediated by astrocytes and microglia could contribute to increased PD susceptibility associated with SARS-CoV-2. These findings advance our understanding of the potential long-term effects of SARS-CoV-2 infection on the progression of PD.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas , Ratones Transgénicos , Enfermedad de Parkinson , SARS-CoV-2 , Animales , Neuronas Dopaminérgicas/patología , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/virología , Humanos , COVID-19/patología , COVID-19/virología , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/virología , Ratones , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Microglía/patología , Microglía/metabolismo , Microglía/virología , Células Madre Embrionarias Humanas/metabolismo , Astrocitos/patología , Astrocitos/virología , Astrocitos/metabolismo , Encéfalo/patología , Encéfalo/virologíaRESUMEN
Parkinson's disease with cognitive impairment (PD-CI) results in several clinical outcomes for which specific treatment is lacking. Although the pathogenesis of PD-CI has not yet been fully elucidated, it is related to neuronal plasticity decline in the hippocampus region. The dopaminergic projections from the substantia nigra to the hippocampus are critical in regulating hippocampal plasticity. Recently, aerobic exercise has been recognized as an effective therapeutic strategy for enhancing plasticity through the secretion of various muscle factors. The exact role of FNDC5-an upregulated, newly identified myokine produced after exercise-in mediating hippocampal plasticity and regional dopaminergic projections in PD-CI remains unclear. In this study, the effect of treadmill exercise on hippocampal synaptic plasticity was evaluated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced chronic PD models. The results showed that treadmill exercise substantially alleviated the motor dysfunction, cognition disorder, and dopaminergic neuron degeneration induced by MPTP. Here, we discovered that the quadriceps, serum, and brain FNDC5 levels were lower in PD mice and that intervention with treadmill exercise restored FNDC5 levels. Moreover, treadmill exercise enhanced the synaptic plasticity of hippocampal pyramidal neurons via increased dopamine levels and BDNF in the PD mice. The direct protective effect of FNDC5 is achieved by promoting the secretion of BDNF in the hippocampal neurons via binding the integrin αVß5 receptor, thereby improving synaptic plasticity. Regarding the indirect protection effect, FNDC5 promotes the dopaminergic connection from the substantia nigra to the hippocampus by mediating the interaction between the integrin αVß5 of the hippocampal neurons and the CD90 molecules on the membrane of dopaminergic terminals. Our findings demonstrated that treadmill exercise could effectively alleviate cognitive disorders via the activation of the FNDC5-BDNF pathway and enhance the dopaminergic synaptic connection from SNpc to the hippocampus in the MPTP-induced chronic PD model.
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Trastornos del Conocimiento , Enfermedad de Parkinson , Ratones , Animales , Enfermedad de Parkinson/metabolismo , Integrina alfaV/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Sustancia Negra/metabolismo , Trastornos del Conocimiento/metabolismo , Dopamina/metabolismo , Factores de Transcripción/metabolismo , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Fibronectinas/metabolismoRESUMEN
Parkinson's disease (PD) is a progressive movement disorder characterized by dopaminergic (DA) neuron degeneration and the existence of Lewy bodies formed by misfolded α-synuclein. Emerging evidence supports the benefits of dietary interventions in PD due to their safety and practicality. Previously, dietary intake of α-ketoglutarate (AKG) was proved to extend the lifespan of various species and protect mice from frailty. However, the mechanism of dietary AKG's effects in PD remains undetermined. In the present study, we report that an AKG-based diet significantly ameliorated α-synuclein pathology, and rescued DA neuron degeneration and impaired DA synapses in adeno-associated virus (AAV)-loaded human α-synuclein mice and transgenic A53T α-synuclein (A53T α-Syn) mice. Moreover, AKG diet increased nigral docosahexaenoic acid (DHA) levels and DHA supplementation reproduced the anti-α-synuclein effects in the PD mouse model. Our study reveals that AKG and DHA induced microglia to phagocytose and degrade α-synuclein via promoting C1q and suppressed pro-inflammatory reactions. Furthermore, results indicate that modulating gut polyunsaturated fatty acid metabolism and microbiota Lachnospiraceae_NK4A136_group in the gut-brain axis may underlie AKG's benefits in treating α-synucleinopathy in mice. Together, our findings propose that dietary intake of AKG is a feasible and promising therapeutic approach for PD.
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Enfermedad de Parkinson , Sinucleinopatías , Ratones , Animales , Humanos , Enfermedad de Parkinson/patología , Ácidos Cetoglutáricos/farmacología , Ratones Transgénicos , Degeneración Nerviosa/patología , Dopamina , Ingestión de Alimentos , Modelos Animales de EnfermedadRESUMEN
Coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) and Coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) are mitochondrial proteins that are thought to be genes which duplicated during evolution and are the causative genes for Parkinson's disease and amyotrophic lateral sclerosis/frontotemporal lobe dementia, respectively. CHCHD2 forms a heterodimer with CHCHD10 and a homodimer with itself, both of which work together within the mitochondria. Various pathogenic and disease-risk variants have been identified; however, how these mutations cause neurodegeneration in specific diseases remains a mystery. This review focuses on important new findings published since 2019 and discusses avenues to solve this mystery.
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Microglia cells are the macrophage population within the central nervous system, which acts as the first line of the immune defense. These cells present a high level of heterogeneity among different brain regions regarding morphology, cell density, transcriptomes, and expression of different inflammatory mediators. This region-specific heterogeneity may lead to different neuroinflammatory responses, influencing the regional involvement in several neurodegenerative diseases. In this study, we aimed to evaluate microglial response in 16 brain regions. We compared different aspects of the microglial response, such as the extension of their morphological changes, sensitivity, and ability to convert an acute inflammatory response to a chronic one. Then, we investigated the synaptic alterations followed by acute and chronic inflammation in substantia nigra. Moreover, we estimated the effect of partial ablation of fractalkine CX3C receptor 1 (CX3CR1) on microglial response. In the end, we briefly investigated astrocytic heterogeneity and activation. To evaluate microglial response in different brain regions and under the same stimulus, we induced a systemic inflammatory reaction through a single intraperitoneal (i.p.) injection of lipopolysaccharides (LPS). We performed our study using C57BL6 and CX3CR1+/GFP mice to investigate microglial response in different regions and the impact of CX3CR1 partial ablation. We conducted a topographic study quantifying microglia alterations in 16 brain regions through immunohistochemical examination and computational image analysis. Assessing Iba1-immunopositive profiles and the density of the microglia cells, we have observed significant differences in region-specific responses of microglia populations in all parameters considered. Our results underline the peculiar microglial inflammation in the substantia nigra pars reticulata (SNpr). Here and in concomitance with the acute inflammatory response, we observed a transient decrease of dopaminergic dendrites and an alteration of the striato-nigral projections. Additionally, we found a significant decrease in microglia response and the absence of chronic inflammation in CX3CR1+/GFP mice compared to the wild-type ones, suggesting the CX3C axis as a possible pharmacological target against neuroinflammation induced by an increase of systemic tumor necrosis factor-alpha (TNFα) or/and LPS. Finally, we investigated astrocytic heterogeneity in this model. We observed different distribution and morphology of GFAP-positive astrocytes, a heterogeneous response under inflammatory conditions, and a decrease in their activation in CX3CR1 partially ablated mice compared with C57BL6 mice. Altogether, our data confirm that microglia and astrocytes heterogeneity lead to a region-specific inflammatory response in presence of a systemic TNFα or/and LPS treatment.
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Microcystins are produced by some species of cyanobacteria, which are hazardous materials to the environment and human beings. It has been demonstrated that microcystin-LR (MC-LR) could disrupt the blood-brain barrier and cause learning and memory deficits, but the neurotoxicity of MC-LR on motor function remains unclear. In this study, the mice were exposed to MC-LR dissolved in drinking water at doses of 1, 7.5, or 15 µg/L for 15 months. We observed that 15 µg/L MC-LR could enter mouse brain tissues such as the cortex, hippocampus, and substantia nigra (SN). And 15 µg/L MC-LR also caused hypokinesia in mice and induced the loss and apoptosis of SN dopaminergic neurons (DA neurons). Meanwhile, MC-LR induced the accumulation of alpha synuclein (α-syn) in DA neurons and decreased the proteins of tyrosine hydroxylase (TH), dopa decarboxylase (DDC) and dopamine transporter (DAT), resulting in a reduction in dopamine (DA) content, which are pathological features of Parkinson's disease (PD). These results suggested that chronic MC-LR might induce PD-like lesions in mice. Moreover, chronic MC-LR exposure caused the inflammatory response in the SN, manifested by the increased numbers of glial cells and the release of inflammatory factors (TNF-α, MCP-1, and IL-6). In vitro, it was proved that MC-LR mediated SH-SY5Y cell apoptosis by activating oxidative stress and damaging mitochondria. Collectively, this study revealed a novel molecular mechanism for MC-LR neurotoxicity with significant implications for human health and the public environment.
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Trastornos del Movimiento , Neuroblastoma , Animales , Humanos , Toxinas Marinas/toxicidad , Ratones , Microcistinas/toxicidadRESUMEN
Sensory neurons enable animals to detect environmental changes and avoid harm. An intriguing open question concerns how the various attributes of sensory neurons arise in development. Drosophila melanogaster larvae undergo a behavioral transition by robustly activating a thermal nociceptive escape behavior during the second half of larval development (third instar). The Class IV dendritic arborization (C4da) neurons are multimodal sensors which tile the body wall of Drosophila larvae and detect nociceptive temperature, light, and mechanical force. In contrast to the increase in nociceptive behavior in the third instar, we find that ultraviolet light-induced Ca2+ activity in C4da neurons decreases during the same period of larval development. Loss of ecdysone receptor has previously been shown to reduce nociception in third instar larvae. We find that ligand-dependent activation of ecdysone signaling is sufficient to promote nociceptive responses in second instar larvae and suppress expression of subdued (encoding a TMEM16 channel). Reduction of subdued expression in second instar C4da neurons not only increases thermal nociception but also decreases the response to ultraviolet light. Thus, steroid hormone signaling suppresses subdued expression to facilitate the sensory switch of C4da neurons. This regulation of a developmental sensory switch through steroid hormone regulation of channel expression raises the possibility that ion channel homeostasis is a key target for tuning the development of sensory modalities.
During their lives, animals encounter a broad range of stimuli from their surroundings including heat, light and touch. The ability to appropriately respond to such stimuli is crucial for survival as it allows the animals to avoid predators and other dangers, locate food and shelter, and find mates. Fruit fly larvae are a useful model for studying how animals respond to unpleasant (known as painful) heat stimuli. When something hot touches a larva, the larva rolls away to avoid the stimulus. The heat stimulates electrical activity in a type of neuron known as C4da neurons on the surface of the larva. Ultraviolet light and several other stimuli are also able to activate electrical activity in C4da neurons, resulting in the larvae changing the direction they move to avoid the stimuli. Only older fly larvae respond to painful heat stimuli and previous studies found that a hormone receptor protein is required for this response. However, it remains unclear how this response develops as the larvae age. Jaszczak et al. studied the behavior of fly larvae and electrical activities of C4da neurons in response to painful heat and ultraviolet light. The experiments found that painful heat triggered more rolling behavior from older larvae than those of younger larvae. In contrast, ultraviolet light triggered lower levels of electrical activity in the C4da neurons of older larvae than those of younger larvae. The team raised the levels of a hormone known as ecdysone and found that this increased the rolling behavior in younger larvae. They then increased the amount of receptor protein for this hormone in the neurons and found that it decreased the levels of another protein called Subdued in the C4da neurons. This in turn increased the neurons' response to painful heat and decreased their response to ultraviolet light. Jaszczak et al. propose that as the larva develops, ecdysone reduces the levels of Subdued, which promotes C4da neurons to switch their sensitivity from detecting ultraviolet light to painful heat. In the future, better understanding of what causes pain sensations in developing animals will help us search for factors that cause long-term pain conditions in humans.
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Drosophila melanogaster , Drosophila , Animales , Drosophila/fisiología , Drosophila melanogaster/metabolismo , Ecdisona/metabolismo , Nocicepción/fisiología , Células Receptoras Sensoriales/metabolismoRESUMEN
The development of efficient cell culture strategies for the generation of dopaminergic neurons is an important goal for transplantation-based approaches to treat Parkinson's disease. To identify extracellular matrix molecules that enhance differentiation and might be used in these cell cultures we have used micro-contact printed arrays on glass slides presenting 190 combinations of 19 extracellular matrix molecules selected on the basis of their expression during embryonic development of the ventral midbrain. Using long-term neuroepithelial stem cells (Lt-NES), this approach identified a number of matricellular proteins that enhanced differentiation, with the combination of Sparc, Sparc-like (Sparc-l1) and Nell2 increasing the number of tyrosine hydroxylase+ neurons derived from Lt-NES cells and, critically for further translation, human pluripotent stem cells.
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The role of the dopaminergic pathway in general anesthesia and its potential mechanisms are still unknown. In this study, we usedc-Fos staining combined with calcium fiber photometry recording to explore the activity of ventral tegmental area (VTA) dopaminergic neurons(VTA-DA) and nucleus accumbens (NAc) neurons during sevoflurane anesthesia. A genetically encoded dopamine (DA) sensor was used to investigate thefunction of the NAc in sevoflurane anesthesia. Chemogenetics and optogenetics were used to explore the role of the VTA-DA in sevofluraneanesthesia. Electroencephalogram (EEG) spectra, time of loss of righting reflex (LORR) and recovery of righting reflex (RORR) were recorded asassessment indicators. We found that VTA-DA and NAc neurons were inhibited during the induction period and were activated during the recoveryperiod of sevoflurane anesthesia. The fluorescence signals of dopamine decreased in the induction of and increased in the emergence from sevoflurane anesthesia.Activation of VTA-DA and the VTADA-NAc pathway delayed the induction and facilitated the emergence accompanying with thereduction of delta band and the augmentation of the gamma band. These data demonstrate that VTA-DA neurons play a critical role in modulating sevofluraneanesthesia via the VTADA-NAc pathway.
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During prolonged nutrient restriction, developing animals redistribute vital nutrients to favor brain growth at the expense of other organs. In Drosophila, such brain sparing relies on a glia-derived growth factor to sustain proliferation of neural stem cells. However, whether other aspects of neural development are also spared under nutrient restriction is unknown. Here we show that dynamically growing somatosensory neurons in the Drosophila peripheral nervous system exhibit organ sparing at the level of arbor growth: Under nutrient stress, sensory dendrites preferentially grow as compared to neighboring non-neural tissues, resulting in dendrite overgrowth. These neurons express lower levels of the stress sensor FoxO than neighboring epidermal cells, and hence exhibit no marked induction of autophagy and a milder suppression of Tor signaling under nutrient stress. Preferential dendrite growth allows for heightened animal responses to sensory stimuli, indicative of a potential survival advantage under environmental challenges.
The organs of a young animal develop in a carefully controlled way to reach the right size relative to each other. However, if the animal's diet does not contain the right amount of nutrients a condition known as malnutrition the body prioritizes the needs of the brain and other vital organs. This means that certain organs keep on growing while others stop. The brain is at the center of the nervous system, which is formed of networks of nerve cells (or neurons) that rapidly carry messages around the body. In the larvae of malnourished fruit flies, a molecular signal allows the nervous system to continue making new neurons as other parts of the body slow down their growth. During development, neurons also connect to each other by growing tree-like structures known as dendrites. However, it remained unclear whether the growth of dendrites was also protected during episodes of malnutrition. To address this question, Poe, Xu et al. performed experiments in the larvae of fruit flies, focusing on a type of neuron whose dendrites extend into the skin. When nutrients were scarce, the neurons grew more rapidly than the surrounding skin cells, resulting in dendrite overgrowth. Compared to neurons, the skin cells had higher levels of a stress sensor known as FoxO, which stops cell growth when nutrients are scarce. Conversely, low quantities of FoxO in neurons allow these cells to keep on growing dendrites, which ultimately helps the starved animals to better react to their environment. These results suggest that the growth of neurons and their connecting structures is preserved during malnutrition. Ultimately, dissecting how organisms prioritize resources can help to develop new approaches to treat human conditions that emerge during malnutrition.
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Dendritas/fisiología , Proteínas de Drosophila/fisiología , Drosophila/crecimiento & desarrollo , Privación de Alimentos , Factores de Transcripción Forkhead/fisiología , Neurogénesis/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Autofagia , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/metabolismo , Células Epidérmicas/fisiología , Femenino , Factores de Transcripción Forkhead/biosíntesis , Sistema de Señalización de MAP Quinasas , Masculino , Nutrientes , Proteínas Tirosina Quinasas Receptoras/metabolismo , Estrés FisiológicoRESUMEN
Objective: To study the therapeutic effect and mechanism of quercetin on Parkinson's disease (PD) model induced by a leucine-rich repeat kinase 2 (LRRK2) gene mutation. Methods: PD transgenic drosophila model Ddc-Gal4; UAS-LRRK2/G2019S was generated by Gal4/UAS hybridization and selectively expressed G2019S mutant LRRK2 in dopaminergic neurons. PD transgenic drosophila and control were fed with corn medium supplemented with quercetin in 1, 10 and 100 μmol/L for the treatment group, or with standard corn medium in PD model group and blank control group. The life span and locomotor ability were observed and compared between the quercetin treatment group and the PD drosophila model group. The brains of the drosophila were dissected and stained with TH immunofluorescence antibody to observe the survival rate of dopaminergic neurons. The brain tissues were also measured with Western blot to detect the protein expression levels of TH, GCLC, p-LRRK2, and p-p38MAPK. Results: The group treated with 10 μmol/L quercetin showed the best therapeutic effect on the prolongation of life span and improvement of locomotor ability compared with PD transgenic drosophila model without any treatment. The locomotor activity of drosophila was significantly improved at week 6 and the loss of dopaminergic neurons in the brain of the PD model drosophila was effectively diminished by quercetin. Quercetin also significantly lowered the level of phosphorylated LRRK2 in the PD transgenic drosophila compared with the PD model group (P<0.05), indicating the inhibiting effect of quercetin on the activity of LRRK2 kinase of the PD model. In addition, quercetin could activate the antioxidant-signaling pathway and inhibit the p38MAPK signaling pathway. Results: Quercetin can activate the antioxidant-signaling pathway and inhibit the LRRK2 kinase activity, which can further regulate MAPK signaling pathway and reduce the neurotoxicity of LRRK2 mutation and protect dopaminergic neurons in PD transgenic drosophila model.
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Building sensory dendritic arbors requires branching, growth, spacing, and substrate support. The conserved L1CAM family of cell-adhesion molecules generates neuronal isoforms to regulate neurite development in various aspects. However, whether non-neuronal isoforms participate in any of these aspects is unclear. In Drosophila, the L1CAM homolog Neuroglian (Nrg) is expressed as two isoforms: the neuronal isoform Nrg180 on dendritic surfaces of dendritic arborization (da) neurons and the non-neuronal isoform Nrg167 in epidermis innervated by dendrites. We found that epidermal Nrg167 encircles dendrites by interactions with dendritic Nrg180 to support dendrite growth, stabilization, and enclosure inside epidermis. Interestingly, whereas Nrg180 forms homophilic interactions to facilitate axonal bundling, heteroneuronal dendrites in the same innervating field avoid bundling through unknown mechanisms to maintain individual dendritic patterns. Here, we show that both epidermal Nrg167 depletion and neuronal Nrg180 overexpression can cause dendrite bundling, with genetic analyses suggesting that Nrg167-Nrg180 interactions antagonize Nrg180-Nrg180 homophilic interaction to prevent dendrite bundling. Furthermore, internalization of Nrg180 also participates in resolving dendrite bundling, as overexpression of endocytosis-defective Nrg180 and compromising endocytosis in neurons both exacerbated dendrite-bundling defects. Together, our study highlights the functional significance of substrate-derived Nrg167 in conferring dendrite stability, positioning, and avoidance.
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Moléculas de Adhesión Celular Neuronal/genética , Dendritas/fisiología , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Isoformas de Proteínas/metabolismoRESUMEN
Sensing environmental cues requires well-built neuronal circuits linked to the body surface. Sensory neurons generate dendrites to innervate surface epithelium, thereby making it the largest sensory organ in the body. Previous studies have illustrated that neuronal type, physiological function and branching patterns are determined by intrinsic factors. Perhaps for effective sensation or protection, sensory dendrites bind to or are surrounded by the substrate epidermis. Recent studies have shed light on the mechanisms by which dendrites interact with their substrates. These interactions suggest that substrates can regulate dendrite guidance, arborization and degeneration. In this review, we focus on recent studies of Drosophila and Caenorhabditis elegans that demonstrate how epidermal cells can regulate dendrites in several aspects.
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Dendritas/metabolismo , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Drosophila/genética , Drosophila/metabolismo , Epidermis/inervación , Larva/genética , Larva/metabolismo , Microscopía Confocal , Imagen de Lapso de Tiempo/métodosRESUMEN
Tissue-specific loss-of-function (LOF) analysis is essential for characterizing gene function. Here, we present a simple, yet highly efficient, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. We describe convenient toolkits for making enhancer-driven Cas9 lines and multi-gRNAs that are optimized for mutagenizing somatic cells. We demonstrate that insertions or deletions in coding sequences more reliably cause somatic mutations than DNA excisions induced by two gRNAs. We further show that enhancer-driven Cas9 is less cytotoxic yet results in more complete LOF than Gal4-driven Cas9 in larval sensory neurons. Finally, CRISPR-TRiM efficiently unmasks redundant soluble N-ethylmaleimide-sensitive factor attachment protein receptor gene functions in neurons and epidermal cells. Importantly, Cas9 transgenes expressed at different times in the neuronal lineage reveal the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISPR tools can be applied to analyze tissue-specific gene function in many biological processes.
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Sistemas CRISPR-Cas , Dosificación de Gen , Técnicas Genéticas , Mutagénesis , Animales , Drosophila/genética , Epidermis/metabolismo , Mutación con Pérdida de Función , Neuronas/metabolismo , Especificidad de Órganos , ARN Guía de Kinetoplastida/genética , TransgenesRESUMEN
BACKGROUND: Parkinson's disease (PD) is caused by the gradual loss of dopamine-producing cells in the brain. This study evaluated the potential neuroprotective role of puerarin (PR) on dopamine (DA)-producing cells in vitro and in vivo. METHOD: In vitro, the effects of PR on proliferation and differentiation and DA releases of mesenchymal stem cells (MSCs) were assayed by CCK-8, flow cytometry, real-time PCR and ELISA respectively. Then the differentiated cells were labeled with enhanced green fluorescent protein (EGFP) and administrated into PD animal models induced by 6-OHDA. The proliferation and differentiation of labeled cells were identified by fluorescence microscopy and immunostaining. RESULTS: In vitro, after being treated with different concentrations of PR for 1 week, the TUJ1, TH and DAT protein and mRNA expression and DA releases increased significantly. In vivo, after transplantation of PR-treated DA-producing cells, the symptoms of PD improved significantly from the second week after transplantation; more transplanted cells survived and migrated to wider region along injection line; more transplanted cells proliferated and differentiated into TH+ cells; more DA was detected in the striatum during 6 weeks' observation. CONCLUSION: The results suggest that PR promote DA neuron survival, proliferation and differentiation.
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Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cuerpo Estriado/cirugía , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/trasplante , Isoflavonas/farmacología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Trastornos Parkinsonianos/cirugía , Animales , Células Cultivadas , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Relación Dosis-Respuesta a Droga , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Oxidopamina , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The potency of mesenchymal stem cells (MSCs) for tissue repair and regeneration is mainly based on their ability to secret beneficial molecules. Administration of MSCs has been proposed as an innovative approach and is proved by a number of clinical trials to a certain degree for the therapy of many diseases including Parkinson's disease (PD). However, the efficacy of MSCs alone is not significant. We investigated the effect of neurotrophic tyrosine receptor kinase 1 (NTRK1) overexpressed peripheral blood MSCs (PB-MSCs) on PD rat model. NTRK1 was overexpressed in PB-MSCs, which were then injected into PD rat model, Dopaminergic (DA) neuron regeneration and rotational performance was assessed. We found that DA neuron repair was increased in lesion site, rotational performance was also improved in MSC transplanted PD rat, with most potent effect in NTRK1 overexpressed PB-MSC transplanted PD rat. Our results indicate that overexpression of NTRK1 in MSCs could be an optimized therapeutic way via MSCs for PD treatment.
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Two recent studies by Meltzer et al. and Ziegler et al. use Drosophila larvae to demonstrate that cell-autonomous regulation of lipid biosynthesis defines the complexity and function of highly branched nociceptive neurons. Their findings show that lipid biosynthesis in the neuron is fine-tuned for optimal dendrite morphology and sensitivity.
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Dendritas , Drosophila , Animales , Proteínas de Drosophila , Larva , Morfogénesis , NeurogénesisRESUMEN
The proper formation of dendritic arbors is a critical step in neural circuit formation, and as such defects in arborization are associated with a variety of neurodevelopmental disorders. Among the best gene candidates are those encoding cell adhesion molecules, including members of the diverse cadherin superfamily characterized by distinctive, repeated adhesive domains in their extracellular regions. Protocadherins (Pcdhs) make up the largest group within this superfamily, encompassing over 80 genes, including the â¼60 genes of the α-, ß-, and γ-Pcdh gene clusters and the non-clustered δ-Pcdh genes. An additional group includes the atypical cadherin genes encoding the giant Fat and Dachsous proteins and the 7-transmembrane cadherins. In this review we highlight the many roles that Pcdhs and atypical cadherins have been demonstrated to play in dendritogenesis, dendrite arborization, and dendritic spine regulation. Together, the published studies we discuss implicate these members of the cadherin superfamily as key regulators of dendrite development and function, and as potential therapeutic targets for future interventions in neurodevelopmental disorders.
Asunto(s)
Cadherinas/fisiología , Dendritas/fisiología , Neuronas/fisiología , Transducción de Señal , Animales , Cadherinas/clasificación , Cadherinas/genética , Moléculas de Adhesión Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Humanos , NeurogénesisRESUMEN
MicroRNA-124 (miR-124) is an evolutionarily conserved, small, noncoding RNA molecule that participates in the central nervous system (CNS) developmental control of gene expression. In the current study, we found that Drosophila embryos lacking the mir-124 gene did not exhibit detectable defects in axon growth or CNS development. On the other hand, adult mutants showed severe problems in locomotion, flight, and female fertility. Furthermore, the deficits that we observed in the adult stage could be marginally rescued with elav-GAL4 driven expression of miR-124, making elav-GAL4 an excellently simulated driver to induce entopic over-expression of miR-124. Further developmental assessment in the third instar larval neuromuscular junction (NMJ) and dendritic arborization (DA) neurons was performed with miR-124 knock outs, flies over-expressing miR-124, and rescue models. Typically, the absence and over-abundance of a molecular signal exerts opposite effects on development or phenotype. However, we determined that both miR-124 knock-outs and over-expressing flies displayed reduced NMJ 6/7 bouton number and branch length. Similarly, reduced ddaE branching numbers were observed between the two mutant lines. As to ddaF, we found that branching number was not influenced by mir-124 knock out, but was statistically reduced by miR-124 over-expression. While we were not able to determine any causal relationship between behavioral defects and dysplasia of NMJs or DA neurons, there were some accompanying relationships among behavioral phenotypes, NMJ abnormalities, and ddaE/ddaF dendritic branching which were all controlled by miR-124.