Tpr Misregulation in Hippocampal Neural Stem Cells in Mouse Models of Alzheimer's Disease.

Nuclear pore complexes (NPCs) are highly dynamic macromolecular protein structures that facilitate molecular exchange across the nuclear envelope. Aberrant NPC functioning has been implicated in neurodegeneration. The translocated promoter region (Tpr) is a critical scaffolding nucleoporin (Nup) of the nuclear basket, facing the interior of the NPC. However, the role of Tpr in adult neural stem/precursor cells (NSPCs) in Alzheimer's disease (AD) is unknown. Using super-resolution (SR) and electron microscopy, we defined the different subcellular localizations of Tpr and phospho-Tpr (P-Tpr) in NSPCs in vitro and in vivo. Elevated Tpr expression and reduced P-Tpr nuclear localization accompany NSPC differentiation along the neurogenic lineage. In 5xFAD mice, an animal model of AD, increased Tpr expression in DCX+ hippocampal neuroblasts precedes increased neurogenesis at an early stage, before the onset of amyloid-ß plaque formation. Whereas nuclear basket Tpr interacts with chromatin modifiers and NSPC-related transcription factors, P-Tpr interacts and co-localizes with cyclin-dependent kinase 1 (Cdk1) at the nuclear chromatin of NSPCs. In hippocampal NSPCs in a mouse model of AD, aberrant Tpr expression was correlated with altered NPC morphology and counts, and Tpr was aberrantly expressed in postmortem human brain samples from patients with AD. Thus, we propose that altered levels and subcellular localization of Tpr in CNS disease affect Tpr functionality, which in turn regulates the architecture and number of NSPC NPCs, possibly leading to aberrant neurogenesis.

Dense dopaminergic innervation of the peri-infarct cortex despite dopaminergic cell loss after a pure motor-cortical stroke in rats.

After ischemic stroke, the cortex directly adjacent to the ischemic core (i.e., the peri-infarct cortex, PIC) undergoes plastic changes that facilitate motor recovery. Dopaminergic signaling is thought to support this process. However, ischemic stroke also leads to the remote degeneration of dopaminergic midbrain neurons, possibly interfering with this beneficial effect. In this study, we assessed the reorganization of dopaminergic innervation of the PIC in a rat model of focal cortical stroke. Adult Sprague-Dawley rats either received a photothrombotic stroke (PTS) in the primary motor cortex (M1) or a sham operation. 30 days after PTS or sham procedure, the retrograde tracer Micro Ruby (MR) was injected into the PIC of stroke animals or into homotopic cortical areas of matched sham rats. Thus, dopaminergic midbrain neurons projecting into the PIC were identified based on MR signal and immunoreactivity against tyrosine hydroxylase (TH), a marker for dopaminergic neurons. The density of dopaminergic innervation within the PIC was assessed by quantification of dopaminergic boutons indicated by TH-immunoreactivity. Regarding postsynaptic processes, expression of dopamine receptors (D1- and D2) and a marker of the functional signal cascade (DARPP-32) were visualized histologically. Despite a 25% ipsilesional loss of dopaminergic midbrain neurons after PTS, the number and spatial distribution of dopaminergic neurons projecting to the PIC was not different compared to sham controls. Moreover, the density of dopaminergic innervation in the PIC was significantly higher than in homotopic cortical areas of the sham group. Within the PIC, D1-receptors were expressed in neurons, whereas D2-receptors were confined to astrocytes. The intensity of D1- and DARPP-32 expression appeared to be higher in the PIC compared to the contralesional homotopic cortex. Our data suggest a sprouting of dopaminergic fibers into the PIC and point to a role for dopaminergic signaling in reparative mechanisms post-stroke, potentially related to recovery.

Fibrinogen regulates lesion border-forming reactive astrocyte properties after vascular damage.

Reactive astrocytes at the border of damaged neuronal tissue organize into a barrier surrounding the fibrotic lesion core, separating this central region of inflammation and fibrosis from healthy tissue. Astrocytes are essential to form the border and for wound repair but interfere with neuronal regeneration. However, the mechanisms driving these astrocytes during central nervous system (CNS) disease are unknown. Here we show that blood-derived fibrinogen is enriched at the interface of lesion border-forming elongated astrocytes after cortical brain injury. Anticoagulant treatment depleting fibrinogen reduces astrocyte reactivity, extracellular matrix deposition and inflammation with no change in the spread of inflammation, whereas inhibiting fibrinogen conversion into fibrin did not significantly alter astrocyte reactivity, but changed the deposition of astrocyte extracellular matrix. RNA sequencing of fluorescence-activated cell sorting-isolated astrocytes of fibrinogen-depleted mice after cortical injury revealed repressed gene expression signatures associated with astrocyte reactivity, extracellular matrix deposition and immune-response regulation, as well as increased gene expression signatures associated with astrocyte metabolism and astrocyte-neuron communication. Systemic pharmacologic depletion of fibrinogen resulted in the absence of elongated, border-forming astrocytes and increased the survival of neurons in the lesion core after cortical injury. These results identify fibrinogen as a critical trigger for lesion border-forming astrocyte properties in CNS disease.

P75 neurotrophin receptor controls subventricular zone neural stem cell migration after stroke.

Stroke is the leading cause of adult disability. Endogenous neural stem/progenitor cells (NSPCs) originating from the subventricular zone (SVZ) contribute to the brain repair process. However, molecular mechanisms underlying CNS disease-induced SVZ NSPC-redirected migration to the lesion area are poorly understood. Here, we show that genetic depletion of the p75 neurotrophin receptor (p75NTR-/-) in mice reduced SVZ NSPC migration towards the lesion area after cortical injury and that p75NTR-/- NSPCs failed to migrate upon BDNF stimulation in vitro. Cortical injury rapidly increased p75NTR abundance in SVZ NSPCs via bone morphogenetic protein (BMP) receptor signaling. SVZ-derived p75NTR-/- NSPCs revealed an altered cytoskeletal network- and small GTPase family-related gene and protein expression. In accordance, BMP-treated non-migrating p75NTR-/- NSPCs revealed an altered morphology and α-tubulin expression compared to BMP-treated migrating wild-type NSPCs. We propose that BMP-induced p75NTR abundance in NSPCs is a regulator of SVZ NSPC migration to the lesion area via regulation of the cytoskeleton following cortical injury.

Id proteins: emerging roles in CNS disease and targets for modifying neural stemcell behavior.

Neural stem/progenitor cells (NSPCs) are found in the adult brain and spinal cord, and endogenous or transplanted NSPCs contribute to repair processes and regulate immune responses in the CNS. However, the molecular mechanisms of NSPC survival and integration as well as their fate determination and functionality are still poorly understood. Inhibitor of DNA binding (Id) proteins are increasingly recognized as key determinants of NSPC fate specification. Id proteins act by antagonizing the DNA-binding activity of basic helix-loop-helix (bHLH) transcription factors, and the balance of Id and bHLH proteins determines cell fate decisions in numerous cell types and developmental stages. Id proteins are central in responses to environmental changes, as they occur in CNS injury and disease, and cellular responses in adult NSPCs implicate Id proteins as prime candidates for manipulating stemcell behavior. Here, we outline recent advances in understanding Id protein pleiotropic functions in CNS diseases and propose an integrated view of Id proteins and their promise as potential targets in modifying stemcell behavior to ameliorate CNS disease.

In vivo functions of p75NTR: challenges and opportunities for an emerging therapeutic target.

The p75 neurotrophin receptor (p75NTR) functions at the molecular nexus of cell death, survival, and differentiation. In addition to its contribution to neurodegenerative diseases and nervous system injuries, recent studies have revealed unanticipated roles of p75NTR in liver repair, fibrinolysis, lung fibrosis, muscle regeneration, and metabolism. Linking these various p75NTR functions more precisely to specific mechanisms marks p75NTR as an emerging candidate for therapeutic intervention in a wide range of disorders. Indeed, small molecule inhibitors of p75NTR binding to neurotrophins have shown efficacy in models of Alzheimer's disease (AD) and neurodegeneration. Here, we outline recent advances in understanding p75NTR pleiotropic functions in vivo, and propose an integrated view of p75NTR and its challenges and opportunities as a pharmacological target.

Fibrin hydrogels promote scar formation and prevent therapeutic angiogenesis in the heart.

Therapeutic angiogenesis is the delivery of factors to promote vascular growth and holds promise for the treatment of ischemic heart conditions. Recombinant protein delivery to the myocardium by factor-decorated fibrin matrices is an attractive approach, thanks to the ability to precisely control both dose and duration of the treatment, the use of a clinically approved material like fibrin, and the avoidance of genetic modification. Here, we investigated the feasibility of inducing therapeutic angiogenesis in the rat myocardium by a state-of-the-art fibrin-based delivery platform that we previously optimized. Engineered versions of murine vascular endothelial growth factor A (VEGF164 ) and platelet-derived growth factor BB (PDGF-BB) were fused with an octapeptide substrate of the transglutaminase coagulation factor fXIIIa (TG) to allow their covalent cross-linking into fibrin hydrogels and release by enzymatic cleavage. Hydrogels containing either 100 µg/mL TG-VEGF alone or in combination with 10 µg/mL TG-PDGF-BB or no factor were injected into rat myocardium. Surprisingly, vascular density was severely reduced in all conditions, both in and around the injection site, where large fibrotic scars were formed. Scar formation was not due to the presence of growth factors, adaptive immunity to human proteins, damage from injection, nor to mechanical trauma from the hydrogel stiffness or volume. Rather scar was induced directly by fibrin and persisted despite hydrogel degradation within 1 week. These results caution against the suitability of fibrin-based platforms for myocardial growth factor delivery, despite their efficacy in other tissues, like skeletal muscle. The underlying molecular mechanisms must be further investigated in order to identify rational targets to prevent this serious side effect.

Fibrinogen induces neural stem cell differentiation into astrocytes in the subventricular zone via BMP signaling.

Neural stem/progenitor cells (NSPCs) originating from the subventricular zone (SVZ) contribute to brain repair during CNS disease. The microenvironment within the SVZ stem cell niche controls NSPC fate. However, extracellular factors within the niche that trigger astrogliogenesis over neurogenesis during CNS disease are unclear. Here, we show that blood-derived fibrinogen is enriched in the SVZ niche following distant cortical brain injury in mice. Fibrinogen inhibited neuronal differentiation in SVZ and hippocampal NSPCs while promoting astrogenesis via activation of the BMP receptor signaling pathway. Genetic and pharmacologic depletion of fibrinogen reduced astrocyte formation within the SVZ after cortical injury, reducing the contribution of SVZ-derived reactive astrocytes to lesion scar formation. We propose that fibrinogen is a regulator of NSPC-derived astrogenesis from the SVZ niche via BMP receptor signaling pathway following injury.

Single-cell RNA-sequencing identifies the developmental trajectory of C-Myc-dependent NK1.1- T-bet+ intraepithelial lymphocyte precursors.

Natural intraepithelial lymphocytes (IELs) are thymus-derived adaptive immune cells, which are important contributors to intestinal immune homeostasis. Similar to other innate-like T cells, they are induced in the thymus through high-avidity interaction that would otherwise lead to clonal deletion in conventional CD4 and CD8 T cells. By applying single-cell RNA-sequencing (scRNA-seq) on a heterogeneous population of thymic CD4-CD8αß-TCRαß+NK1.1- IEL precursors (NK1.1- IELPs), we define a developmental trajectory that can be tracked based on the sequential expression of CD122 and T-bet. Moreover, we identify the Id proteins Id2 and Id3 as a novel regulator of IELP development and show that all NK1.1- IELPs progress through a PD-1 stage that precedes the induction of T-bet. The transition from PD-1 to T-bet is regulated by the transcription factor C-Myc, which has far reaching effects on cell cycle, energy metabolism, and the translational machinery during IELP development. In summary, our results provide a high-resolution molecular framework for thymic IEL development of NK1.1- IELPs and deepen our understanding of this still elusive cell type.

The Concerted Action of E2-2 and HEB Is Critical for Early Lymphoid Specification.

The apparition of adaptive immunity in Gnathostomata correlates with the expansion of the E-protein family to encompass E2-2, HEB, and E2A. Within the family, E2-2 and HEB are more closely evolutionarily related but their concerted action in hematopoiesis remains to be explored. Here we show that the combined disruption of E2-2 and HEB results in failure to express the early lymphoid program in Common lymphoid precursors (CLPs) and a near complete block in B-cell development. In the thymus, Early T-cell progenitors (ETPs) were reduced and T-cell development perturbed, resulting in reduced CD4 T- and increased γδ T-cell numbers. In contrast, hematopoietic stem cells (HSCs), erythro-myeloid progenitors, and innate immune cells were unaffected showing that E2-2 and HEB are dispensable for the ancestral hematopoietic lineages. Taken together, this E-protein dependence suggests that the appearance of the full Gnathostomata E-protein repertoire was critical to reinforce the gene regulatory circuits that drove the emergence and expansion of the lineages constituting humoral immunity.

TGFß-Signaling and FOXG1-Expression Are a Hallmark of Astrocyte Lineage Diversity in the Murine Ventral and Dorsal Forebrain.

Heterogeneous astrocyte populations are defined by diversity in cellular environment, progenitor identity or function. Yet, little is known about the extent of the heterogeneity and how this diversity is acquired during development. To investigate the impact of TGF (transforming growth factor) ß-signaling on astrocyte development in the telencephalon we deleted the TGFBR2 (transforming growth factor beta receptor 2) in early neural progenitor cells in mice using a FOXG1 (forkhead box G1)-driven CRE-recombinase. We used quantitative proteomics to characterize TGFBR2-deficient cells derived from the mouse telencephalon and identified differential protein expression of the astrocyte proteins GFAP (glial fibrillary acidic protein) and MFGE8 (milk fat globule-EGF factor 8). Biochemical and histological investigations revealed distinct populations of astrocytes in the dorsal and ventral telencephalon marked by GFAP or MFGE8 protein expression. The two subtypes differed in their response to TGFß-signaling. Impaired TGFß-signaling affected numbers of GFAP astrocytes in the ventral telencephalon. In contrast, TGFß reduced MFGE8-expression in astrocytes deriving from both regions. Additionally, lineage tracing revealed that both GFAP and MFGE8 astrocyte subtypes derived partly from FOXG1-expressing neural precursor cells.

Regulatory T cells characterized by low Id3 expression are highly suppressive and accumulate during chronic infection.

Foxp3+ regulatory T (Treg) cells are broadly divided into naive-like and activated Treg cells, however recent studies suggest further Treg cell heterogeneity. Treg cells contribute to impaired T cell responses in chronic infections, but the role of specific Treg cell subpopulations in viral infections is not well defined. Here, we report that activated Treg cells are separated into two transcriptionally distinct subpopulations characterized by low or high expression of the transcriptional regulator Id3. Id3lo Treg cells are a highly suppressive Treg cell subpopulation, expressing elevated levels of immunomodulatory molecules and are capable of broadly targeting T cell responses. Viral infection and interleukin-2 promote the differentiation of Id3hi into Id3lo Treg cells and during chronic infection Id3lo Treg cells are the predominant Treg cell population. Thus, our report provides a framework, in which different activated Treg cell subpopulations specifically affect immune responses, possibly contributing to T cell dysfunction in chronic infections.

Fibrinogen Activates BMP Signaling in Oligodendrocyte Progenitor Cells and Inhibits Remyelination after Vascular Damage.

Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived molecules serve as extrinsic inhibitors of remyelination is unknown. Here we show that the coagulation factor fibrinogen activates the bone morphogenetic protein (BMP) signaling pathway in oligodendrocyte progenitor cells (OPCs) and suppresses remyelination. Fibrinogen induces phosphorylation of Smad 1/5/8 and inhibits OPC differentiation into myelinating oligodendrocytes (OLs) while promoting an astrocytic fate in vitro. Fibrinogen effects are rescued by BMP type I receptor inhibition using dorsomorphin homolog 1 (DMH1) or CRISPR/Cas9 activin A receptor type I (ACVR1) knockout in OPCs. Fibrinogen and the BMP target Id2 are increased in demyelinated multiple sclerosis (MS) lesions. Therapeutic depletion of fibrinogen decreases BMP signaling and enhances remyelination in vivo. Targeting fibrinogen may be an upstream therapeutic strategy to promote the regenerative potential of CNS progenitors in diseases with remyelination failure.

The E2A splice variant E47 regulates the differentiation of projection neurons via p57(KIP2) during cortical development.

During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ventricular and subventricular zones, from where they migrate towards the pial surface to assemble into highly organized layer-specific circuits. However, the precise and coordinated transcriptional network activity defining neuronal identity is still not understood. Here, we show that genetic depletion of the basic helix-loop-helix (bHLH) transcription factor E2A splice variant E47 increased the number of Tbr1-positive deep layer and Satb2-positive upper layer neurons at E14.5, while depletion of the alternatively spliced E12 variant did not affect layer-specific neurogenesis. While ChIP-Seq identified a big overlap for E12- and E47-specific binding sites in embryonic NSCs, including sites at the cyclin-dependent kinase inhibitor (CDKI) Cdkn1c gene locus, RNA-Seq revealed a unique transcriptional regulation by each splice variant. E47 activated the expression of the CDKI Cdkn1c through binding to a distal enhancer. Finally, overexpression of E47 in embryonic NSCs in vitro impaired neurite outgrowth, and overexpression of E47 in vivo by in utero electroporation disturbed proper layer-specific neurogenesis and upregulated p57(KIP2) expression. Overall, this study identifies E2A target genes in embryonic NSCs and demonstrates that E47 regulates neuronal differentiation via p57(KIP2).

Id3 Maintains Foxp3 Expression in Regulatory T Cells by Controlling a Transcriptional Network of E47, Spi-B, and SOCS3.

The transcription factor Foxp3 dominantly controls regulatory T (Treg) cell function, and only its continuous expression guarantees the maintenance of full Treg cell-suppressive capacity. However, transcriptional regulators maintaining Foxp3 transcription are incompletely described. Here, we report that high E47 transcription factor activity in Treg cells resulted in unstable Foxp3 expression. Under homeostatic conditions, Treg cells expressed high levels of the E47 antagonist Id3, thus restricting E47 activity and maintaining Foxp3 expression. In contrast, stimulation of Id3-deficient or E47-overexpressing Treg cells resulted in the loss of Foxp3 expression in a subset of Treg cells in vivo and in vitro. Mechanistic analysis indicated that E47 activated expression of the transcription factor Spi-B and the suppressor of cytokine signaling 3 (SOCS3), which both downregulated Foxp3 expression. These findings demonstrate that the balance of Id3 and E47 controls the maintenance of Foxp3 expression in Treg cells and, thus, contributes to Treg cell plasticity.

ID(ealizing) control of adult subventricular zone neural stem/precursor cell differentiation for CNS regeneration.

The adult central nervous system (CNS) was considered a comparatively static tissue with little cell turnover. It is now well established that there is more plasticity than previously thought and that astrocytes act as neural stem/precursor cells (NSPCs) in the subventricular zone (SVZ). The discovery that these NSPCs can give rise to a limited number of new neurons, reactive astrocytes and oligodendrocytes contributing to brain repair in CNS disease, has raised hopes toward harnessing these cells for therapeutic interventions. Here, we will discuss the transcriptional control of adult NSPC differentiation into astrocytes in CNS disease focusing on the helix-loop-helix transcription factor protein family. In our recent study, we reported that elevated BMP-2 levels are translated into an increase in Id3 expression in adult NSPC subpopulations after cortical injury. Id3 then heterodimerizes with the basic helix-loop-helix transcription factor E47 and releases the E47-mediated repression of astrocyte-specific gene expression. Consequently, adult NSPCs preferentially differentiate into astrocytes. We believe that understanding the in vivo differentiation potential and the molecular underpinnings of NSPCs in the adult mammalian brain will help us to evaluate their contributions to brain repair and may lead to new concepts in treating human CNS diseases.

The balance of Id3 and E47 determines neural stem/precursor cell differentiation into astrocytes.

Adult neural stem/precursor cells (NSPCs) of the subventricular zone (SVZ) are an endogenous source for neuronal replacement in CNS disease. However, adult neurogenesis is compromised after brain injury in favor of a glial cell fate, which is mainly attributed to changes in the NSPC environment. Yet, it is unknown how this unfavorable extracellular environment translates into a transcriptional program altering NSPC differentiation. Here, we show that genetic depletion of the transcriptional regulator Id3 decreased the number of astrocytes generated from SVZ-derived adult NSPCs in the cortical lesion area after traumatic brain injury. Cortical brain injury resulted in rapid BMP-2 and Id3 up-regulation in the SVZ stem cell niche. Id3(-/-) adult NSPCs failed to differentiate into BMP-2-induced astrocytes, while NSPCs deficient for the Id3-controlled transcription factor E47 readily differentiated into astrocytes in the absence of BMP-2. Mechanistically, E47 repressed the expression of several astrocyte-specific genes in adult NSPCs. These results identify Id3 as the BMP-2-induced transcriptional regulator, promoting adult NSPC differentiation into astrocytes upon CNS injury and reveal a molecular link between environmental changes and NSPC differentiation in the CNS after injury.