systems-analysis-neuroinflammatory-hemodynamic-response-to-traumatic

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

Georgia Institute of Technology

The development of bone requires carefully choregraphed signaling to bone progenitors to form bone. Our group recently described the requirement of transforming growth factor beta receptor 3 (TGFβR3), a receptor involved in TGFβ pathway signaling, during osteoblast lineage commitment in mice. The TGFβ pathway is known to play multiple osteo-inductive and osteo-inhibitory roles during osteoblast development and TGFβR3 human mutations are associated with reduced bone mineral density, making TGFβR3 a unique target for bone inductive therapy. In this article, we demonstrated increased mineralization of human pediatric bone-derived osteoblast-like cells (HBO) when treated with soluble TGFβR3 (sR3) using Alizarin Red staining. Osteogenic commitment of HBO cells was demonstrated by induction of osteogenic genes RUNX2, osteocalcin, osteopontin, and osterix. Evaluation of the canonical TGFβ pathway signaling demonstrated that sR3 was able to induce bone formation in HBO cells, mainly through activation of noncanonical targets of TGFβ pathway signaling including AKT, ERK, and p38 MAP kinases. Inhibition of these osteogenic noncanonical pathways in the HBO cells also inhibited mineralization, suggesting they are each required. Although no induction of SMAD1, 5, and 9 was observed, there was the activation of SMAD2 and 3 suggesting that sR3 is primarily signaling via the noncanonical pathways during osteogenic induction of the HBO. Our results highlight the important role of TGFβR3 in osteoblast induction of mineralization in human bone cells through noncanonical targets of TGFβ signaling. Future studies will focus on the ability of sR3 to induce bone regeneration in vivo using animal models.

Osteoinductive effect of soluble transforming growth factor beta receptor 3 on human osteoblast lineage.

Kv1.3 potassium channels, expressed by proinflammatory central nervous system mononuclear phagocytes (CNS-MPs), are promising therapeutic targets for modulating neuroinflammation in Alzheimer's disease (AD). The molecular characteristics of Kv1.3-high CNS-MPs and their cellular origin from microglia or CNS-infiltrating monocytes are unclear. While Kv1.3 blockade reduces amyloid beta (Aβ) burden in mouse models, the downstream immune effects on molecular profiles of CNS-MPs remain unknown. We show that functional Kv1.3 channels are selectively expressed by a subset of CD11bCD45 CNS-MPs acutely isolated from an Aβ mouse model (5xFAD) as well as fresh postmortem human AD brain. Transcriptomic profiling of purified CD11bKv1.3 CNS-MPs, CD11bCD45 Kv1.3 microglia, and peripheral monocytes from 5xFAD mice revealed that Kv1.3-high CNS-MPs highly express canonical microglial markers (, ) and are distinct from peripheral Ly6c/Ly6c monocytes. Unlike homeostatic microglia, Kv1.3-high CNS-MPs express relatively lower levels of homeostatic genes, higher levels of , and increased levels of glutamatergic transcripts, potentially representing phagocytic uptake of neuronal elements. Using irradiation bone marrow CD45.1/CD45.2 chimerism in 5xFAD mice, we show that Kv1.3 CNS-MPs originate from microglia and not blood-derived monocytes. We show that Kv1.3 channels regulate membrane potential and early signaling events in microglia. Finally, in vivo blockade of Kv1.3 channels in 5xFAD mice by ShK-223 reduced Aβ burden, increased CD11c CNS-MPs, and expression of phagocytic genes while suppressing proinflammatory genes (). Our results confirm the microglial origin and identify unique molecular features of Kv1.3-expressing CNS-MPs. In addition, we provide evidence for CNS immunomodulation by Kv1.3 blockers in AD mouse models resulting in a prophagocytic phenotype.

Unique molecular characteristics and microglial origin of Kv1.3 channel-positive brain myeloid cells in Alzheimer's disease.

The BIN1 locus contains the second-most significant genetic risk factor for late-onset Alzheimer's disease. BIN1 undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer's disease, have not been examined in depth.

BIN1 is a key regulator of proinflammatory and neurodegeneration-related activation in microglia.

Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We describe a mouse line for cell type-specific expression of biotin ligase TurboID, for in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show robust protein biotinylation in neuronal soma and axons throughout the brain, allowing quantitation of over 2000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we contrast Camk2a-neuron and Aldh1l1-astrocyte proteomes and identify brain region-specific proteomic differences within both cell types, some of which might potentially underlie the selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we apply an antibody-based approach to uncover differences in neuron and astrocyte-derived signaling phospho-proteins and cytokines. This approach will facilitate the characterization of cell-type specific proteomes in a diverse number of tissues under both physiological and pathological states.

Article	Year
Osteoinductive effect of soluble transforming growth factor beta receptor 3 on human osteoblast lineage. Journal of cellular biochemistry\| PubMed ID: 33480071	2021
Unique molecular characteristics and microglial origin of Kv1.3 channel-positive brain myeloid cells in Alzheimer's disease. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 33649184	2021
BIN1 is a key regulator of proinflammatory and neurodegeneration-related activation in microglia. Molecular neurodegeneration\| PubMed ID: 35526014	2022
Cell type-specific biotin labeling in vivo resolves regional neuronal and astrocyte proteomic differences in mouse brain. Nature communications\| PubMed ID: 35614064	2022

Sara Bitarafan

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