Molecular and Cellular Neuroscience最新文献

Microglia are widely known for their role in immune surveillance and for their ability to refine neurocircuitry during development, but a growing body of evidence suggests that microglia may also play a complementary role to neurons in regulating the behavioral aspects of substance use disorders. While many of these efforts have focused on changes in microglial gene expression associated with drug-taking, epigenetic regulation of these changes has yet to be fully understood. This review provides recent evidence supporting the role of microglia in various aspects of substance use disorder, with particular focus on changes to the microglial transcriptome and the potential epigenetic mechanisms driving these changes. Further, this review discusses the latest technical advances in low-input chromatin profiling and highlights the current challenges for studying these novel molecular mechanisms in microglia.

Drugs of abuse increase extracellular concentrations of dopamine in the nucleus accumbens (NAc), resulting in transcriptional alterations that drive long-lasting cellular and behavioral adaptations. While decades of research have focused on the transcriptional mechanisms by which drugs of abuse influence neuronal physiology and function, few studies have comprehensively defined NAc cell type heterogeneity in transcriptional responses to drugs of abuse. Here, we used single nucleus RNA-seq (snRNA-seq) to characterize the transcriptome of over 39,000 NAc cells from male and female adult Sprague-Dawley rats following acute or repeated cocaine experience. This dataset identified 16 transcriptionally distinct cell populations, including two populations of medium spiny neurons (MSNs) that express the Drd1 dopamine receptor (D1-MSNs). Critically, while both populations expressed classic marker genes of D1-MSNs, only one population exhibited a robust transcriptional response to cocaine. Validation of population-selective transcripts using RNA in situ hybridization revealed distinct spatial compartmentalization of these D1-MSN populations within the NAc. Finally, analysis of published NAc snRNA-seq datasets from non-human primates and humans demonstrated conservation of MSN subtypes across rat and higher order mammals, and further highlighted cell type-specific transcriptional differences across the NAc and broader striatum. These results highlight the utility in using snRNA-seq to characterize both cell type heterogeneity and cell type-specific responses to cocaine and provides a useful resource for cross-species comparisons of NAc cell composition.

The endoplasmic reticulum (ER) is the largest membrane compartment within eukaryotic cells and is emerging as a key coordinator of many cellular processes. The ER can modulate local calcium fluxes and communicate with other organelles like the plasma membrane. The importance of ER in neuronal processes such as neurite growth, axon repair and neurotransmission has recently gained much attention. In this review, we highlight the importance of the ER tubular network in axonal homeostasis and discuss how the generation and maintenance of the thin tubular ER network in axons and synapses, requires a cooperative effort of ER-shaping proteins, cytoskeleton and autophagy processes.

Brain derived neurotrophic factor (BDNF) signalling through its high-affinity tropomyosin receptor kinase B (TrkB) is known to have potent effects on motor neuron survival and morphology during development and in neurodegenerative diseases. Here, we employed a novel 1NMPP1 sensitive TrkB^F616 rat model to evaluate the effect of 14 days inhibition of TrkB signalling on phrenic motor neurons (PhMNs). Adult female and male TrkB^F616 rats were divided into 1NMPP1 or vehicle treated groups. Three days prior to treatment, PhMNs in both groups were initially labeled via intrapleural injection of Alexa-Fluor-647 cholera toxin B (CTB). After 11 days of treatment, retrograde axonal uptake/transport was assessed by secondary labeling of PhMNs by intrapleural injection of Alexa-Fluor-488 CTB. After 14 days of treatment, the spinal cord was excised 100 μm thick spinal sections containing PhMNs were imaged using two-channel confocal microscopy. TrkB inhibition reduced the total number of PhMNs by ∼16 %, reduced the mean PhMN somal surface areas by ∼25 %, impaired CTB uptake 2.5-fold and reduced the estimated PhMN dendritic surface area by ∼38 %. We conclude that inhibition of TrkB signalling alone in adult TrkB^F616 rats is sufficient to lead to PhMN loss, morphological degeneration and deficits in retrograde axonal uptake/transport.

During Alzheimer's (AD), tau protein suffers from abnormal post-translational modifications, including cleaving by caspase-3. These tau forms affect synaptic plasticity contributing to the cognitive decline observed in the early stages of AD. In addition, caspase-3 cleaved tau (TauC3) impairs mitochondrial dynamics and organelles transport, which are both relevant processes for synapse. We recently showed that the absence of tau expression reverts age-associated cognitive and mitochondrial failure by blocking the mitochondrial permeability transition pore (mPTP). mPTP is a mitochondrial complex involved in calcium regulation and apoptosis. Therefore, we studied the effects of TauC3 against the dendritic spine and synaptic vesicle formation and the possible role of mPTP in these alterations. We used mature hippocampal mice neurons to express a reporter protein (GFP, mCherry), coupled to full-length human tau protein (GFP-T4, mCherry-T4), and coupled to human tau protein cleaved at D421 by caspase-3 (GFP-T4C3, mCherry-T4C3) and synaptic elements were evaluated.

Treatment with cyclosporine A (CsA), an immunosuppressive drug with inhibitory activity on mPTP, prevented ROS increase and mitochondrial depolarization induced by TauC3 in hippocampal neurons. These results were corroborated with immortalized cortical neurons in which ROS increase and ATP loss induced by this tau form were prevented by CsA. Interestingly, TauC3 expression significantly reduced dendritic spine density (filopodia type) and synaptic vesicle number in hippocampal neurons. Also, neurons transfected with TauC3 showed a significant accumulation of synaptophysin protein in their soma. More importantly, all these synaptic alterations were prevented by CsA, suggesting an mPTP role in these negative changes derived from TauC3 expression.

Traumatic brain injury is a leading cause of morbidity and mortality in adults and children in developed nations. Following the primary injury, microglia, the resident innate immune cells of the CNS, initiate several inflammatory signaling cascades and pathophysiological responses that may persist chronically; chronic neuroinflammation following TBI has been closely linked to the development of neurodegeneration and neurological dysfunction. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been shown to regulate several key mechanisms in the inflammatory response to TBI. Increasing evidence has shown that the modulation of the PI3K/AKT signaling pathway has the potential to influence the cellular response to inflammatory stimuli. However, directly targeting PI3K signaling poses several challenges due to its regulatory role in several cell survival pathways. We have previously identified that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), the major negative regulator of PI3K/AKT signaling, is dysregulated following exposure to repetitive mild traumatic brain injury (r-mTBI). Moreover, this dysregulated PI3K/AKT signaling was correlated with chronic microglial-mediated neuroinflammation. Therefore, we interrogated microglial-specific PTEN as a therapeutic target in TBI by generating a microglial-specific, Tamoxifen inducible conditional PTEN knockout model using a CX3CR1 Cre recombinase mouse line PTEN^fl/fl/CX3CR1^+/CreERT2 (mcg-PTEN^cKO), and exposed them to our 20-hit r-mTBI paradigm. Animals were treated with tamoxifen at 76 days post-last injury, and the effects of microglia PTEN deletion on immune-inflammatory responses were assessed at 90-days post last injury. We observed that the deletion of microglial PTEN ameliorated the proinflammatory response to repetitive brain trauma, not only reducing chronic microglial activation and proinflammatory cytokine production but also rescuing TBI-induced reactive astrogliosis, demonstrating that these effects extended beyond microglia alone. Additionally, we observed that the pharmacological inhibition of PTEN with BpV(HOpic) ameliorated the LPS-induced activation of microglial NFκB signaling in vitro. Together, these data provide support for the role of PTEN as a regulator of chronic neuroinflammation following repetitive mild TBI.

Tardigrades are microscopic invertebrates, which are capable of withstanding extreme environmental conditions, including high levels of radiation. A Tardigrade protein, Dsup (Damage Suppressor), protects the Tardigrade's DNA during harsh environmental stress and X-rays. When expressed in cancer cells, Dsup protects DNA from single- and double-strand breaks (DSBs) induced by radiation, increases survival of irradiated cells, and protects DNA from reactive oxygen species. These unusual properties of Dsup suggested that understanding how the protein functions may help in the design of small molecules that could protect humans during radiotherapy or space travel. Here, we investigated if Dsup is protective in cortical neurons cultured from rat embryos. We discovered that, in cortical neurons, the codon-optimized Dsup localizes to the nucleus and, surprisingly, promotes neurotoxicity, leading to neurodegeneration. Unexpectedly, we found that Dsup expression results in the formation of DNA DSBs in cultured neurons. With electron microscopy, we discovered that Dsup promotes chromatin condensation. Unlike Dsup's protective properties in cancerous cells, in neurons, Dsup promotes neurotoxicity, induces DNA damage, and rearranges chromatin. Neurons are sensitive to Dsup, and Dsup is a doubtful surrogate for DNA protection in neuronal cells.

Drug addiction is a leading cause of disability worldwide, with more than 70,000 Americans dying from drug overdose in 2019 alone. While only a small percentage of chronic drug users escalate to drug addiction, little is understood on the precise mechanisms of this susceptibility. Early life adversity is causally relevant to adult psychiatric disease and may contribute to the risk of addiction. Here we review recent pre-clinical evidence showing that early life exposure to stress and/or drugs regulates changes in behavior, gene expression, and the epigenome that persist into adulthood. We summarize the major findings and gaps in the preclinical literature, highlighting studies that demonstrate the often profound differences between female and male subjects.

Polymorphisms and altered expression of the Translocase of the Outer Mitochondrial Membrane – 40 kD (Tom40) are observed in neurodegenerative disease subjects. We utilized in vitro cultured dorsal root ganglion (DRG) neurons to investigate the association of TOM40 depletion to neurodegeneration, and to unravel the mechanism of neurodegeneration induced by decreased levels of TOM40 protein. We provide evidence that severity of neurodegeneration induced in the TOM40 depleted neurons increases with the increase in the depletion of TOM40 and is exacerbated by an increase in the duration of TOM40 depletion. We also demonstrate that TOM40 depletion causes a surge in neuronal calcium levels, decreases mitochondrial motility, increases mitochondrial fission, and decreases neuronal ATP levels. We observed that alterations in the neuronal calcium homeostasis and mitochondrial dynamics precede BCL-xl and NMNAT1 dependent neurodegenerative pathways in the TOM40 depleted neurons. This data also suggests that manipulation of BCL-xl and NMNAT1 may be of therapeutic value in TOM40 associated neurodegenerative disorders.

High turnover rates of synaptic proteins imply that synapses constantly need to replace their constituent building blocks. This requires sophisticated supply chains and potentially exposes synapses to shortages as they compete for limited resources. Interestingly, competition in neurons has been observed at different scales. Whether it is competition of receptors for binding sites inside a single synapse or synapses fighting for resources to grow. Here we review the implications of such competition for synaptic function and plasticity. We identify multiple mechanisms that synapses use to safeguard themselves against supply shortages and identify a fundamental neurologistic trade-off governing the sizes of reserve pools of essential synaptic building blocks.