Central Nervous System Cell Types Research Articles

Delivery of Adeno-Associated Virus Vectors to the Central Nervous System for Correction of Single Gene Disorders.

Genetic disorders of the central nervous system (CNS) comprise a significant portion of disability in both children and adults. Several preclinical animal models have shown effective adeno-associated virus (AAV) mediated gene transfer for either treatment or prevention of autosomal recessive genetic disorders. Owing to the intricacy of the human CNS and the blood-brain barrier, it is difficult to deliver genes, particularly since the expression of any given gene may be required in a particular CNS structure or cell type at a specific time during development. In this review, we analyzed delivery methods for AAV-mediated gene therapy in past and current clinical trials. The delivery routes analyzed were direct intraparenchymal (IP), intracerebroventricular (ICV), intra-cisterna magna (CM), lumbar intrathecal (IT), and intravenous (IV). The results demonstrated that the dose used in these routes varies dramatically. The average total doses used were calculated and were 1.03 × 1013 for IP, 5.00 × 1013 for ICV, 1.26 × 1014 for CM, and 3.14 × 1014 for IT delivery. The dose for IV delivery varies by patient weight and is 1.13 × 1015 IV for a 10 kg infant. Ultimately, the choice of intervention must weigh the risk of an invasive surgical procedure to the toxicity and immune response associated with a high dose vector.

A fluorescent splice-switching mouse model enables high-throughput, sensitive quantification of antisense oligonucleotide delivery and activity

While antisense oligonucleotides (ASOs) are used in the clinic, therapeutic development is hindered by the inability to assay ASO delivery and activity invivo. Accordingly, we developed a dual-fluorescence, knockin mouse model that constitutively expresses mKate2 and an engineered EGFP that is alternatively spliced in the presence of ASO to induce expression. We first examined free ASO activity in the brain following intracerebroventricular injection revealing EGFP splice-switching is both ASO concentration and time dependent in major central nervous system cell types. We then assayed the impact of lipid nanoparticle delivery on ASO activity after intravenous administration. Robust EGFP fluorescence was observed in the liver and EGFP+ cells were successfully isolated using fluorescence-activated cell sorting. Together, these results show the utility of this animal model in quantifying both cell-type- and organ-specific ASO delivery, which can be used to advance ASO therapeutics for many disease indications.

TUNEL-n-DIFL Method for Detection and Estimation of Apoptosis Specifically in Neurons and Glial Cells in Mixed Culture and Animal Models of Central Nervous System Diseases and Injuries.

Detection of merely apoptosis does not reveal the type of central nervous system (CNS) cells that are dying in the CNS diseases and injuries. In situ detection and estimation of amount of apoptosis specifically in neurons or glial cells (astrocytes, oligodendrocytes, and microglia) can unveil valuable information for designing therapeutics for protection of the CNS cells and functional recovery. A method was first developed and reported from our laboratory for in situ detection and estimation of amount of apoptosis precisely in neurons and glial cells using in vitro and in vivo models of CNS diseases and injuries. This is a combination of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and double immunofluorescent labeling (DIFL) or simply TUNEL-n-DIFL method for in situ detection and estimation of amount of apoptosis in aspecific CNS cell type. An anti-digoxigenin (DIG) IgG antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) for blue fluorescence, fluorescein isothiocyanate (FITC) for green fluorescence, or Texas Red (TR) for red fluorescence can be used for in situ detection of apoptotic cell DNA, which is earlier labeled with TUNEL using alkali-stable DIG-11-dUTP. A primary anti-NeuN (neurons), anti-GFAP (astrocytes), anti-MBP (oligodendrocytes), or anti-OX-42 (microglia) IgG antibody and a secondary IgG antibody conjugated with one of the above fluorophores (other than that of ani-DIG antibody) are used for in situ detection of apoptosis in a specific CNS celltype in the mixed culture and animal models of the CNS diseases and injuries.

Evaluating dynamic autophagic responses to tau inclusions

AbstractBackgroundAlzheimer’s disease and other tauopathies are characterised by the accumulation of misfolded microtubule‐associated protein tau in intracellular inclusions. We recently showed that newly formed tau inclusions are dynamic structures with a slower, but constant turnover of aggregated tau in murine organotypic brain slice cultures (BSCs) (Croft et al., 2021). Clearance by autophagy or the proteasomal machinery are indicated to be the key mechanisms involved in pathogenic protein clearance. However, the current tools to measure these processes have received mixed review, and in particular struggle to capture these processes in a continuous manner.MethodsTo dynamically assess autophagy in the presence of tau inclusions we have repurposed the autophagic flux sensor (GFP‐LC3‐RFP∆G‐LC3) to be delivered by rAAV to specific central nervous system (CNS) cell types in BSCs. This probe delivers a cytosolic internal control so autophagic flux can be estimated by calculating the GFP/RFP ratio. When utilized in conjunction with live imaging, the rate of autophagic flux can be measured over time in the same CNS cells expressing pathological or physiological tau. The rate of autophagic flux can also be measured in response to established/novel compounds or genetic manipulation.ResultsWe have successfully developed and characterized the use of rAAV‐GFP‐LC3‐RFP∆G‐LC3 specifically in neurons and astrocytes in BSCs and are expanding its use to other CNS cell types. We have validated its use to sense autophagic flux whilst pharmacologically enhancing and inhibiting autophagy in BSCs. We are now using this sensor to determine dynamic autophagic processes in the presence of soluble tau and aggregated tau and its response to other manipulations.ConclusionsMethods to understand autophagic processes have traditionally only provided snapshots over time. With this tool we can now dynamically assess autophagy in ex vivo BSCs and characterize changes in response to tau and other pathologies. Overall, understanding the mechanisms that enable tau turnover may guide therapies for Alzheimer’s disease and other neurodegenerative conditions where tau plays a role. Importantly, we will also begin to understand whether enhancing tau inclusion clearance may be beneficial or detrimental to cell health.

Apolipoprotein E polymorphism‐dependent effects on cellular function

AbstractBackgroundWe have used a combination of cell‐based and biophysical approaches to investigate mechanisms by which apolipoprotein E (APOE) isoforms induce either protective or Alzheimer’s Disease (AD)‐associated pathological features.MethodAPOE polymorphism‐driven phenotypic and functional changes were investigated in both primary human astrocytes and central nervous system (CNS) cell types generated from a set of APOE isogenic hiPSC lines (APOE KO, E2, E3, E4). Using fluorescently‐labeled APOE, the transport of E2, E3 and E4 isoforms were tracked in live cells and changes in organelle co‐localization was assessed. Lipoprotein particles were isolated from conditioned media to investigate APOE polymorphism‐specific alternations in the assembly and secretion of high‐density lipoprotein (HDL) particles.ResultWe have identified APOE polymorphism‐specific changes in subcellular localization of APOE that are correlated with changes in cellular function, as well as the function of APOE as a major transporter of lipids and cholesterol in the brain.ConclusionOur results provide mechanistic insight into structure:function relationship of APOE in the context of pathological and protective isoforms.

Migraine and major depression: localizing shared genetic susceptibility in different cell types of the nervous systems.

There is a bidirectional relationship between migraine and major depression disorder (MDD). They likely share important risk genes associated with different cell types in the central nervous system (CNS) and peripheral nervous system (PNS). Profiling the expression of these genes in specific cell types is critical in understanding the pathophysiology of the relationship between migraine and MDD. Associated genes shared by migraine and MDD were identified by consolidating multiple curations of human disease-gene associations. Subsequently, the expression of overlapping genes was profiled and compared across the different cell types in CNS, PNS and neurovascular cells using eight single cell RNA sequencing datasets, including two human CNS datasets, two mouse CNS datasets, one human PNS dataset and three mouse PNS datasets. 45 shared genes between migraine and MDD were identified. Consistently found in all eight datasets, dopaminergic and serotonergic neurotransmitters were broadly expressed in CNS and PNS cell types. Glutamatergic and endocannabinoid genes were specifically expressed in CNS neurons and astrocytes. Synthesis and/or Release and Binding of Neuropeptides were specifically expressed in PNS peptidergic nociceptor (PEP). Genes related to inflammatory factors and immune responses were specifically expressed in CNS microglia. Among which, IL1B and COMT were highly expressed in CNS microglia cells. Single cell RNA sequencing of the CNS and PNS helps to identify the shared genes between migraine and MDD that are enriched in specific cell types. The findings provide new insight in understanding the underlying mechanism of action for the bidirectional co-morbidity between migraine and MDD.

The versatile applications of human pluripotent stem cell-derived microglia and microglia-containing brain organoids

Microglia are the resident immune cells of the central nervous system (CNS) and play pivotal roles in nervous development, homeostasis, and various neurological diseases. Most of the previous understanding of microglia came from rodents or a limited number of postmortem microglia. However, as significant differences between murine and human microglia have been verified, it has become increasingly apparent that rodents cannot accurately recapitulate human genetics and pathology, thus hindering the translation of microglial findings from rodents to humans. In addition, primary human microglia are notoriously difficult to obtain and lack the scalability required for many high-throughput assays. Fortunately, recent advances in microglia generation from human pluripotent stem cells (hPSCs) have enabled exciting new avenues to decipher or revisit microglial biology in the human context. Given the complex interactions between microglia and other CNS cells, hPSC-derived microglia-like cells (MGLs) were further engrafted within hPSC-derived brain organoids (BOs), which largely lack microglia due to their different embryonic origins, to study human microglial functions in either health and disease state closer to brain microglia. This is a rapidly evolving field, especially in the last five years, that has begun to yield novel insights into the genetics of human microglia and their unique role in neurological diseases. In this review, we will summarize the versatile applications of hPSC-derived MGLs and microglia-containing BOs. Specifically, we will discuss their applications in disease modeling, omics and systematic analysis, interaction with other CNS cell types, as well as transplantation-based human-mouse chimerism.

Loss of Function in the Neurodevelopmental Disease and Schizophrenia-Associated Gene CYFIP1 in Human Microglia-like Cells Supports a Functional Role in Synaptic Engulfment

BackgroundThe CYFIP1 gene, located in the neurodevelopmental risk locus 15q11.2, is highly expressed in microglia, but its role in human microglial function as it relates to neurodevelopment is not well understood. MethodsWe generated multiple CRISPR (clustered regularly interspaced short palindromic repeat) knockouts of CYFIP1 in patient-derived models of microglia to characterize function and phenotype. Using microglia-like cells reprogrammed from peripheral blood mononuclear cells, we quantified phagocytosis of synaptosomes (isolated and purified synaptic vesicles) from human induced pluripotent stem cell (iPSC)–derived neuronal cultures as an in vitro model of synaptic pruning. We repeated these analyses in human iPSC-derived microglia-like cells derived from 3 isogenic wild-type/knockout line pairs derived from 2 donors and further characterized microglial development and function through morphology and motility. ResultsCYFIP1 knockout using orthogonal CRISPR constructs in multiple patient-derived cell lines was associated with a statistically significant decrease in synaptic vesicle phagocytosis in microglia-like cell models derived from both peripheral blood mononuclear cells and iPSCs. Morphology was also shifted toward a more ramified profile, and motility was significantly reduced. However, iPSC-CYFIP1 knockout lines retained the ability to differentiate to functional microglia. ConclusionsThe changes in microglial phenotype and function due to the loss of function of CYFIP1 observed in this study implicate a potential impact on processes such as synaptic pruning that may contribute to CYFIP1-related neurodevelopmental disorders. Investigating risk genes in a range of central nervous system cell types, not solely neurons, may be required to fully understand the way in which common and rare variants intersect to yield neuropsychiatric disorders.

In silico-in vitro modeling to uncover cues involved in establishing microglia identity: TGF-β3 and laminin can drive microglia signature gene expression.

Microglia are the resident macrophages of the central nervous system (CNS) and play a key role in CNS development, homeostasis, and disease. Good in vitro models are indispensable to study their cellular biology, and although much progress has been made, in vitro cultures of primary microglia still only partially recapitulate the transcriptome of in vivo microglia. In this study, we explored a combination of in silico and in vitro methodologies to gain insight into cues that are involved in the induction or maintenance of the ex vivo microglia reference transcriptome. First, we used the in silico tool NicheNet to investigate which (CNS-derived) cues could underlie the differences between the transcriptomes of ex vivo and in vitro microglia. Modeling on basis of gene products that were found to be upregulated in vitro, predicted that high mobility group box 2 (HMGB2)- and interleukin (IL)-1β-associated signaling pathways were driving their expression. Modeling on basis of gene products that were found to be downregulated in vitro, did not lead to predictions on the involvement of specific signaling pathways. This is consistent with the idea that in vivo microenvironmental cues that determine microglial identity are for most part of inhibitory nature. In a second approach, primary microglia were exposed to conditioned medium from different CNS cell types. Conditioned medium from spheres composed of microglia, oligodendrocytes, and radial glia, increased the mRNA expression levels of the microglia signature gene P2RY12. NicheNet analyses of ligands expressed by oligodendrocytes and radial glia predicted transforming growth factor beta 3 (TGF-β3) and LAMA2 as drivers of microglia signature gene expression. In a third approach, we exposed microglia to TGF-β3 and laminin. In vitro exposure to TGF-β3 increased the mRNA expression levels of the microglia signature gene TREM2. Microglia cultured on laminin-coated substrates were characterized by reduced mRNA expression levels of extracellular matrix-associated genes MMP3 and MMP7, and by increased mRNA expression levels of the microglia signature genes GPR34 and P2RY13. Together, our results suggest to explore inhibition of HMGB2- and IL-1β-associated pathways in in vitro microglia. In addition, exposure to TGF-β3 and cultivation on laminin-coated substrates are suggested as potential improvements to current in vitro microglia culture protocols.

A single-cell map of antisense oligonucleotide activity in the brain.

Antisense oligonucleotides (ASOs) dosed into cerebrospinal fluid (CSF) distribute broadly throughout the central nervous system (CNS). By modulating RNA, they hold the promise of targeting root molecular causes of disease and hold potential to treat myriad CNS disorders. Realization of this potential requires that ASOs must be active in the disease-relevant cells, and ideally, that monitorable biomarkers also reflect ASO activity in these cells. The biodistribution and activity of such centrally delivered ASOs have been deeply characterized in rodent and non-human primate (NHP) models, but usually only in bulk tissue, limiting our understanding of the distribution of ASO activity across individual cells and across diverse CNS cell types. Moreover, in human clinical trials, target engagement is usually monitorable only in a single compartment, CSF. We sought a deeper understanding of how individual cells and cell types contribute to bulk tissue signal in the CNS, and how these are linked to CSF biomarker outcomes. We employed single nucleus transcriptomics on tissue from mice treated with RNase H1 ASOs against Prnp and Malat1 and NHPs treated with an ASO against PRNP. Pharmacologic activity was observed in every cell type, though sometimes with substantial differences in magnitude. Single cell RNA count distributions implied target RNA suppression in every single sequenced cell, rather than intense knockdown in only some cells. Duration of action up to 12 weeks post-dose differed across cell types, being shorter in microglia than in neurons. Suppression in neurons was generally similar to, or more robust than, the bulk tissue. In macaques, PrP in CSF was lowered 40% in conjunction with PRNP knockdown across all cell types including neurons, arguing that a CSF biomarker readout is likely to reflect ASO pharmacodynamic effect in disease-relevant cells in a neuronal disorder. Our results provide a reference dataset for ASO activity distribution in the CNS and establish single nucleus sequencing as a method for evaluating cell type specificity of oligonucleotide therapeutics and other modalities.

Conserved and cell type-specific transcriptional responses to IFN-γ in the ventral midbrain

Dysregulated inflammation within the central nervous system (CNS) contributes to neuropathology in infectious, autoimmune, and neurodegenerative disease. With the exception of microglia, major histocompatibility complex (MHC) proteins are virtually undetectable in the mature, healthy central nervous system (CNS). Neurons have generally been considered incapable of antigen presentation, and although interferon gamma (IFN-γ) can elicit neuronal MHC class I (MHC-I) expression and antigen presentation in vitro, it has been unclear whether similar responses occur in vivo. Here we directly injected IFN-γ into the ventral midbrain of mature mice and analyzed gene expression profiles of specific CNS cell types. We found that IFN-γ upregulated MHC-I and associated mRNAs in ventral midbrain microglia, astrocytes, oligodendrocytes, and GABAergic, glutamatergic, and dopaminergic neurons. The core set of IFN-γ-induced genes and their response kinetics were similar in neurons and glia, but with a lower amplitude of expression in neurons. A diverse repertoire of genes was upregulated in glia, particularly microglia, which were the only cells to undergo cellular proliferation and express MHC classII (MHC-II) and associated genes. To determine if neurons respond directly via cell-autonomous IFN-γ receptor (IFNGR) signaling, we produced mutant mice with a deletion of the IFN-γ-binding domain of IFNGR1 in dopaminergic neurons, which resulted in a complete loss of dopaminergic neuronal responses to IFN-γ. Our results demonstrate that IFN-γ induces neuronal IFNGR signaling and upregulation of MHC-I and related genes in vivo, although the expression level is low compared to oligodendrocytes, astrocytes, and microglia.

Issue Information

Front cover The cellular expression and functions of the sigma-1 receptor (S1R) in the central nervous system (CNS) still remain elusive due to a lack of appropriate tools. For the first time since its discovery over forty years ago, the sigma-1 receptor was specifically detected in all central nervous system (CNS) cell types in vivo by a newly established immunohistochemical protocol. We took advantage of S1R knockout mice to identify a commercial antibody (Ab#61994) specifically recognizing S1Rs in immunoblots. We further established a simple immunohistochemical antigen retrieval method employing 1% SDS to reliably detect S1Rs with Ab#61994 in brain vibratome sections, demonstrating a wide expression of S1R in neurons and all glial cell types. We also generated a new S1R flox mouse for Cre-dependent conditional deletion of S1Rs for functional analysis in vivo. Image content Brain sections from wild-type (WT) and S1R knockout (KO) mice were pre-treated with 1% SDS and immunostained by various S1R antibodies separately. Scale bars = 200 μm. Read the full article ‘Specific detection and deletion of the sigma-1 receptor widely expressed in neurons and glial cells in vivo’ by Q. Liu, Q. Guo, L-P. Fang, H. Yao, A. Scheller, F. Kirchhoff, W. Huang, (J. Neurochem. 2023, vol.164 (6), pp. 764–785) on doi:10.1111/jnc.15693

The Epidemiology of Brain and Spinal Cord Tumors.

CNS tumors are a diverse group of neoplasms that emerge from a variety of different CNS cell types. These tumors may be benign, malignant, or borderline in nature. The majority of high grade glial tumors are fatal, with the exception of pilocytic astrocytoma. Primary malignant CNS tumors occur at a global annual rate of 2.1 to 5.8 per 100,000 persons. Males are more likely to develop malignant brain tumors than females, whereas benign meningiomas are more common in adultfemales. Additionally, gender inequalities in non-malignant tumors peak between the ages of 25 and 29years. Only a small number of genetic variants have been associated with survival and prognosis. Notably, central nervous system (CNS) tumors exhibit significant age, gender, and race variation. Race is another factor that affects the incidence of brain and spinal cord tumors. Different races exhibit variation in terms of the prevalence of brain and CNS malignancies. This chapter discusses ongoing research on brain and spinal cord tumor epidemiology, as well as the associated risks and accompanied disorders.

Exercise-induced modulation of neuroinflammation in ageing.

Optimal performance of the central nervous system (CNS) depends on dynamic, multidirectional communication between different cell types both within and without the CNS to maintain the homeostatic environment. Ageing, in turn, is associated with CNS disequilibrium resulting in suboptimal functioning of its cells and potential cognitive impairment. Emerging evidence indicates that inter-organ communication influences the functioning of CNS cell types, which are subject to age- and environment-dependent alterations. Endurance exercise has specifically been demonstrated to have a marked impact on neuroimmune communications, particularly those involving microglia, the resident macrophages of the CNS parenchyma, as well as microglia-astrocyte interactions in rodents. Via its action on CNS glial cells, regular aerobic exercise has been shown to provide an adaptive advantage against perturbations to homeostasis, such as immunological challenge or ageing. In light of the accumulating evidence and evolutionary reasoning it may be argued that recurrent exercise-associated inter-organ signalling is necessary for the optimisation of glial function and hence CNS equilibrium. This, in turn, would imply that the absence of exercise-derived mediators and dysregulated inter-organ communication associated with a sedentary lifestyle may contribute to CNS dyshomeostasis, which is accelerated during ageing. As well as exploring the evidence of the impact of exercise on glial function, here we suggest potential next steps in identifying the mechanistic underpinnings of these effects and the potential importance of sex differences.

Spatially resolved transcriptomics in the APPSWE [Tg2576] mouse model of Alzheimer’s disease

AbstractBackgroundAlzheimer's and other neurodegenerative diseases remain pervasive, impacting patients and their families as the disease progresses. Despite the continual expansion of our understanding of these conditions, the detailed molecular mechanisms and progression of pathology throughout the brain remain elusive and complex. Using genomic and RNA‐seq approaches, identification of individual cells and their genetic makeup provide an added perspective on their roles in how the central nervous system (CNS) function in both healthy and diseased states. Here we demonstrate the characterization of spatial transcriptomic patterns in the APPSWE [Tg2576] mouse model of familial Alzheimer’s Disease using the Visium Spatial Gene Expression Solution.MethodSerial sections of fresh‐frozen transgenic APPSWE [Tg2576] and littermate control mouse brains were placed onto Visium Spatial Gene Expression slides. The slides consist of ∼5,000 spot arrays with uniquely barcoded probes allowing for the capture of native mRNA. The samples were H&E or immunostained and then permeabilized, allowing for the capture of transcripts and library generation. The resulting libraries were then sequenced on an Illumina NovaSeq. Then, using the 10x Genomics Space Ranger analysis pipeline and the Loupe Browser desktop software, the RNA‐seq data were merged with stained tissue images to align reads, perform clustering, and gene expression analysis.ResultWith the generated data, we demonstrated an improved version of the spotted oligo array technology that increases tissue coverage and spatial resolution with reduced spot size, and increased spot number and packing density. We captured spatial patterns of gene expression and mapped the information back to H&E and immunostained images with regional annotations. Performing differential analysis on the resulting spatial gene expression data, we observed elevated expression of the Pmch (pro‐melanin concentrating hormone) gene within the hypothalamic region of APPSWE [Tg2576] transgenic mice compared to the littermate controls. By immunofluorescence staining, we confirmed the gene expression patterns and utilized this approach to identify the cells and refine the gene expression characterization.ConclusionTogether, this confluence of imaging and sequencing is a valuable tool for understanding the relationships between CNS cell types in healthy and diseased samples with an unbiased anatomical and gene expression‐driven method.

Pyroptosis in spinal cord injury.

Spinal cord injury (SCI) often brings devastating consequences to patients and their families. Pathophysiologically, the primary insult causes irreversible damage to neurons and glial cells and initiates the secondary damage cascade, further leading to inflammation, ischemia, and cells death. In SCI, the release of various inflammatory mediators aggravates nerve injury. Pyroptosis is a new pro-inflammatory pattern of regulated cell death (RCD), mainly mediated by caspase-1 or caspase-11/4/5. Gasdermins family are pore-forming proteins known as the executor of pyroptosis and the gasdermin D (GSDMD) is best characterized. Pyroptosis occurs in multiple central nervous system (CNS) cell types, especially plays a vital role in the development of SCI. We review here the evidence for pyroptosis in SCI, and focus on the pyroptosis of different cells and the crosstalk between them. In addition, we discuss the interaction between pyroptosis and other forms of RCD in SCI. We also summarize the therapeutic strategies for pyroptosis inhibition, so as to provide novel ideas for improving outcomes following SCI.

Antiviral response within different cell types of the CNS.

The central nervous system (CNS) is a constitutive structure of various cell types conserved by anatomical barriers. Many of the major CNS cell-type populations distributed across the different brain regions are targets for several neurotropic viruses. Numerous studies have demonstrated that viral susceptibility within the CNS is not absolute and initiates a cell-type specific antiviral defence response. Neurons, astrocytes, and microglial cells are among the major resident cell populations within the CNS and are all equipped to sense viral infection and induce a relative antiviral response mostly through type I IFN production, however, not all these cell types adopt a similar antiviral strategy. Rising evidence has suggested a diversity regarding IFN production and responsiveness based on the cell type/sub type, regional distinction and cell`s developmental state which could shape distinct antiviral signatures. Among CNS resident cell types, neurons are of the highest priority to defend against the invading virus due to their poor renewable nature. Therefore, infected and uninfected glial cells tend to play more dominant antiviral roles during a viral infection and have been found to be the major CNS IFN producers. Alternatively, neuronal cells do play an active part during antiviral responses but may adopt differential strategies in addition to induction of a typical type I IFN response, to minimize the chance of cellular damage. Heterogeneity observed in neuronal IFN responsiveness may be partially explained by their altered ISGs and/or lower STATS expression levels, however, further in vivo studies are required to fully elucidate the specificity of the acquired antiviral responses by distinct CNS cell types.

Ketogenic diet uncovers differential metabolic plasticity of brain cells.

To maintain homeostasis, the body, including the brain, reprograms its metabolism in response to altered nutrition or disease. However, the consequences of these challenges for the energy metabolism of the different brain cell types remain unknown. Here, we generated a proteome atlas of the major central nervous system (CNS) cell types from young and adult mice, after feeding the therapeutically relevant low-carbohydrate, high-fat ketogenic diet (KD) and during neuroinflammation. Under steady-state conditions, CNS cell types prefer distinct modes of energy metabolism. Unexpectedly, the comparison with KD revealed distinct cell type–specific strategies to manage the altered availability of energy metabolites. Astrocytes and neurons but not oligodendrocytes demonstrated metabolic plasticity. Moreover, inflammatory demyelinating disease changed the neuronal metabolic signature in a similar direction as KD. Together, these findings highlight the importance of the metabolic cross-talk between CNS cells and between the periphery and the brain to manage altered nutrition and neurological disease.

Central nervous system cholesterol metabolism in health and disease.

Cholesterol is a ubiquitous and essential component of cellular membranes, as it regulates membrane structure and fluidity. Furthermore, cholesterol serves as a precursor for steroid hormones, oxysterol, and bile acids, that are essential for maintaining many of the body's metabolic processes. The biosynthesis and excretion of cholesterol is tightly regulated in order to maintain homeostasis. Although virtually all cells have the capacity to make cholesterol, the liver and brain are the two main organs producing cholesterol in mammals. Once produced, cholesterol is transported in the form of lipoprotein particles to other cell types and tissues. Upon formation of the blood-brain barrier (BBB) during embryonic development, lipoproteins cannot move between the central nervous system (CNS) and the rest of the body. As such, cholesterol biosynthesis and metabolism in the CNS operate autonomously without input from the circulation system in normal physiological conditions. Nevertheless, similar regulatory mechanisms for maintaining cholesterol homeostasis are utilized in both the CNS and peripheral systems. Here, we discuss the functions and metabolism of cholesterol in the CNS. We further focus on how different CNS cell types contribute to cholesterol metabolism, and how ApoE, the major CNS apolipoprotein, is involved in normal and pathophysiological functions. Understanding these basic mechanisms will aid our ability to elucidate how CNS cholesterol dysmetabolism contributes to neurogenerative diseases.

Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.