Astrocytes play critical roles after brain injury, but their precise function is poorly defined. Utilizing single-nuclei transcriptomics to characterize astrocytes after ischemic stroke in the visual ...cortex of the marmoset monkey, we observed nearly complete segregation between stroke and control astrocyte clusters. Screening for the top 30 differentially expressed genes that might limit stroke recovery, we discovered that a majority of astrocytes expressed RTN4A/ NogoA, a neurite-outgrowth inhibitory protein previously only associated with oligodendrocytes. NogoA upregulation on reactive astrocytes post-stroke was significant in both the marmoset and human brain, whereas only a marginal change was observed in mice. We determined that NogoA mediated an anti-inflammatory response which likely contributes to limiting the infiltration of peripheral macrophages into the surviving parenchyma.
Aging is the greatest risk factor for neurodegeneration, but the connection between the two processes remains opaque. This is in part for want of a rigorous way to define physiological age, as ...opposed to chronological age. Here, we develop a comprehensive metric for physiological age in
, based on genome-wide expression profiling. We applied this metric to a model of adult-onset neurodegeneration, increased or decreased expression of the activating subunit of the Cdk5 protein kinase, encoded by the gene
, the ortholog of mammalian p35.
-mediated degeneration was associated with a 27-150% acceleration of the intrinsic rate of aging, depending on the tissue and genetic manipulation. Gene ontology analysis and direct experimental tests revealed that affected age-associated processes included numerous core phenotypes of neurodegeneration, including enhanced oxidative stress and impaired proteostasis. Taken together, our results suggest that
-mediated neurodegeneration results from accelerated aging, in combination with cell-autonomous neuronal insults. These data fundamentally recast our picture of the relationship between neurodegeneration and its most prominent risk factor, natural aging.
Progression of Alzheimer's disease leads to synapse loss, neural network dysfunction and cognitive failure. Accumulation of protein aggregates and brain immune activation have triggering roles in ...synaptic failure but the neuronal mechanisms underlying synapse loss are unclear. On the neuronal surface, cellular prion protein (PrP
) is known to be a high-affinity binding site for Amyloid-β oligomers (Aβo). However, PrP
's dependence in knock-in AD models for tau accumulation, transcriptomic alterations and imaging biomarkers is unknown.
The necessity of PrP
was examined as a function of age in homozygous App
/hMapt double knock-in mice (DKI). Phenotypes of App
/hMapt mice with a deletion of Prnp expression (DKI; Prnp
) were compared with DKI mice with intact Prnp, mice with a targeted deletion of Prnp (Prnp
), and mice with intact Prnp (WT). Phenotypes examined included behavioral deficits, synapse loss by PET imaging, synapse loss by immunohistology, tau pathology, gliosis, inflammatory markers, and snRNA-seq transcriptomic profiling.
By 9 months age, DKI mice showed learning and memory impairment, but DKI; Prnp
and Prnp
groups were indistinguishable from WT. Synapse loss in DKI brain, measured by 18FSynVesT-1 SV2A PET or anti-SV2A immunohistology, was prevented by Prnp deletion. Accumulation of Tau phosphorylated at aa 217 and 202/205, C1q tagging of synapses, and dystrophic neurites were all increased in DKI mice but each decreased to WT levels with Prnp deletion. In contrast, astrogliosis, microgliosis and Aβ levels were unchanged between DKI and DKI; Prnp
groups. Single-nuclei transcriptomics revealed differential expression in neurons and glia of DKI mice relative to WT. For DKI; Prnp
mice, the majority of neuronal genes differentially expressed in DKI mice were no longer significantly altered relative to WT, but most glial DKI-dependent gene expression changes persisted. The DKI-dependent neuronal genes corrected by Prnp deletion associated bioinformatically with synaptic function. Additional genes were uniquely altered only in the Prnp
or the DKI; Prnp
groups.
Thus, PrP
-dependent synapse loss, phospho-tau accumulation and neuronal gene expression in AD mice can be reversed without clearing Aβ plaque or preventing gliotic reaction. This supports targeting the Aβo-PrP
interaction to prevent Aβo-neurotoxicity and pathologic tau accumulation in AD.
Synapse loss is one of the hallmarks of Alzheimer's disease (AD) and is associated with cognitive decline. In this study, we tested
FSDM-16, a novel metabolically stable SV2A PET imaging probe, in ...the transgenic APPswe/PS1dE9 (APP/PS1) mouse model of AD and age-matched wild-type (WT) mice at 12 months of age.
Based on previous preclinical PET imaging studies using
CUCB-J and
FSynVesT-1 in the same strain animals, we used the simplified reference tissue model (SRTM), with brain stem as the pseudo reference region to calculate distribution volume ratios (DVRs).
To simplify and streamline the quantitative analysis, we compared the standardized uptake value ratios (SUVRs) from different imaging windows to DVRs and found that the averaged SUVRs from 60-90 min post-injection (
.) are most consistent with the DVRs. Thus, we used averaged SUVRs from 60-90 min for group comparisons and found statistically significant differences in the tracer uptake in different brain regions, e.g., hippocampus (
= 0.001), striatum (
= 0.002), thalamus (
= 0.003), and cingulate cortex (
= 0.0003).
In conclusion,
FSDM-16 was used to detect decreased SV2A levels in the brain of APP/PS1 AD mouse model at one year old. Our data suggest that
FSDM-16 has similar statistical power in detecting the synapse loss in APP/PS1 mice as
CUCB-J and
FSynVesT-1, albeit later imaging window (60-90 min
.) is needed when SUVR is used as a surrogate for DVR for
FSDM-16 due to its slower brain kinetics.
Human and Simian Immunodeficiency virus (HIV-1, HIV-2, and SIV) encode an accessory protein, Nef, which is a pathogenesis and virulence factor. Nef is a multivalent adapter that dysregulates the ...trafficking of many immune cell receptors, including chemokine receptors (CKRs). Physiological endocytic itinerary of agonist occupied CXCR4 involves ubiquitinylation of the phosphorylated receptor at three critical lysine residues and dynamin-dependent trafficking through the ESCRT pathway into lysosomes for degradation. Likewise, Nef induced CXCR4 degradation was critically dependent on the three lysines in the C-terminal -SSLKILSKGK- motif. Nef directly recruits the HECT domain E3 ligases AIP4 or NEDD4 to CXCR4 in the resting state. This mechanism was confirmed by ternary interactions of Nef, CXCR4 and AIP4 or NEDD4; by reversal of Nef effect by expression of catalytically inactive AIP4-C830A mutant; and siRNA knockdown of AIP4, NEDD4 or some ESCRT-0 adapters. However, ubiquitinylation dependent lysosomal degradation was not the only mechanism by which Nef downregulated CKRs. Agonist and Nef mediated CXCR2 (and CXCR1) degradation was ubiquitinylation independent. Nef also profoundly downregulated the naturally truncated CXCR4 associated with WHIM syndrome and engineered variants of CXCR4 that resist CXCL12 induced internalization via an ubiquitinylation independent mechanism.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gene rearrangement occurs during development in some cell types and this genome dynamics is modulated by intrinsic and extrinsic factors, including growth stimulants and nutrients. This raises a ...possibility that such structural change in the genome and its subsequent epigenetic modifications may also take place during mammalian ontogeny, a process undergoing finely orchestrated cell division and differentiation. We tested this hypothesis by comparing single nucleotide polymorphism-defined haplotype frequencies and DNA methylation of the rDNA multicopy gene between two mouse ontogenic stages and among three adult tissues of individual mice. Possible influences to the genetic and epigenetic dynamics by paternal exposures were also examined for Cr(III) and acid saline extrinsic factors. Variables derived from litters, individuals, and duplicate assays in large mouse populations were examined using linear mixed-effects model. We report here that active rDNA rearrangement, represented by changes of haplotype frequencies, arises during ontogenic progression from day 8 embryos to 6-week adult mice as well as in different tissue lineages and is modifiable by paternal exposures. The rDNA methylation levels were also altered in concordance with this ontogenic progression and were associated with rDNA haplotypes. Sperm showed highest level of methylation, followed by lungs and livers, and preferentially selected haplotypes that are positively associated with methylation. Livers, maintaining lower levels of rDNA methylation compared with lungs, expressed more rRNA transcript. In vitro transcription demonstrated haplotype-dependent rRNA expression. Thus, the genome is also dynamic during mammalian ontogeny and its rearrangement may trigger epigenetic changes and subsequent transcriptional controls, that are further influenced by paternal exposures.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or ...solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in
Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His
6-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.
Prostate cancers of luminal adenocarcinoma histology display a range of clinical behaviors. Although most prostate cancers are slow-growing and indolent, a proportion is aggressive, developing ...metastasis and resistance to androgen deprivation treatment. One hypothesis is that a portion of aggressive cancers initiate from stem-like, androgen-independent tumor-propagating cells. Here we demonstrate the in vitro creation of a mouse cell line, selected for growth as self-renewing stem/progenitor cells, which manifests many in vivo properties of aggressive prostate cancer. Normal mouse prostate epithelium containing floxed Pten and TP53 alleles was subjected to CRE-mediated deletion in vitro followed by serial propagation as protospheres. A polyclonal cell line was established from dissociated protospheres and subsequently a clonal daughter line was derived. Both lines demonstrate a mature luminal phenotype in vitro. The established lines contain a stable minor population of progenitor cells with protosphere-forming ability and multi-lineage differentiation capacity. Both lines formed orthotopic adenocarcinoma tumors with metastatic potential to lung. Intracardiac inoculation resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the bone metastatic phenotype of human prostate cancer. The cells showed androgen receptor dependent growth in vitro. Importantly, in vivo, the deprivation of androgens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. These data suggest that transformed prostate progenitor cells preferentially differentiate toward luminal cells and recapitulate many characteristics of the human disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Innate immunity is central to the pathophysiology of neurodegenerative disorders, but it remains unclear why immunity is altered in the disease state and whether changes in immunity are a cause or a ...consequence of neuronal dysfunction. Here, we identify a molecular pathway that links innate immunity to age-dependent loss of dopaminergic neurons in Drosophila. We find, first, that altering the expression of the activating subunit of the Cdk5 protein kinase (Cdk5α) causes severe disruption of autophagy. Second, this disruption of autophagy is both necessary and sufficient to cause the hyperactivation of innate immunity, particularly expression of anti-microbial peptides. Finally, it is the upregulation of immunity that induces the age-dependent death of dopaminergic neurons. Given the dysregulation of Cdk5 and innate immunity in human neurodegeneration and the conserved role of the kinase in the regulation of autophagy, this sequence is likely to have direct application to the chain of events in human neurodegenerative disease.
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•Altering Cdk5 activity causes degeneration of dopamine neurons in Drosophila•Increasing or decreasing Cdk5 activity impairs autophagic flux•Impairing autophagic flux hyperactivates innate immunity•The hyperactivity of immunity, in turn, causes the age-dependent neuron loss
How can one disentangle the many pathologies of neurodegeneration from one another and from normal aging? Shukla et al. show that a mutation in Drosophila kills neurons by impairing autophagy, which in turn stimulates neurotoxic levels of innate immunity, and this acts synergistically with a parallel pathway that accelerates aging.
The hippocampal-entorhinal system supports cognitive functions, has lifelong neurogenic capabilities in many species, and is selectively vulnerable to Alzheimer’s disease. To investigate neurogenic ...potential and cellular diversity, we profiled single-nucleus transcriptomes in five hippocampal-entorhinal subregions in humans, macaques, and pigs. Integrated cross-species analysis revealed robust transcriptomic and histologic signatures of neurogenesis in the adult mouse, pig, and macaque but not humans. Doublecortin (DCX), a widely accepted marker of newly generated granule cells, was detected in diverse human neurons, but it did not define immature neuron populations. To explore species differences in cellular diversity and implications for disease, we characterized subregion-specific, transcriptomically defined cell types and transitional changes from the three-layered archicortex to the six-layered neocortex. Notably, METTL7B defined subregion-specific excitatory neurons and astrocytes in primates, associated with endoplasmic reticulum and lipid droplet proteins, including Alzheimer’s disease-related proteins. This resource reveals cell-type- and species-specific properties shaping hippocampal-entorhinal neurogenesis and function.
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•Single-nucleus RNA-seq of adult hippocampal-entorhinal cells in human, monkey, and pig•Transcriptomic signatures of adult neurogenesis in mouse, pig, and monkey but not human•Excitatory neuron diversification delineates transitions from 3- to 6-layered cortex•METTL7B defines subregion-specific excitatory neurons and astrocytes in primates
Using snRNA-seq of the adult human, macaque, and pig hippocampal-entorhinal system, Franjic et al. defined shared and divergent cell type features, like primate-specific expression of METTL7B in some excitatory neurons and astrocytes. They also identified robust transcriptomic and histologic signatures of neurogenesis in the mouse, pig, and macaque but not humans.
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