Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1β

Abstract Background The cause of neurodegeneration in progressive forms of multiple sclerosis is unknown. We investigated the impact of specific neuroinflammatory markers on human neurons to identify potential therapeutic targets for neuroprotection against chronic inflammation. Methods Surface immunocytochemistry directly visualized protease-activated receptor-1 (PAR1) and interleukin-1 (IL-1) receptors on neurons in human postmortem cortex in patients with and without neuroinflammatory lesions. Viability of cultured neurons was determined after exposure to cerebrospinal fluid from patients with progressive multiple sclerosis or purified granzyme B and IL-1β. Inhibitors of PAR1 activation and of PAR1-associated second messenger signaling were used to elucidate a mechanism of neurotoxicity. Results Immunohistochemistry of human post-mortem brain tissue demonstrated cells expressing higher amounts of PAR1 near and within subcortical lesions in patients with multiple sclerosis compared to control tissue. Human cerebrospinal fluid samples containing granzyme B and IL-1β were toxic to human neuronal cultures. Granzyme B was neurotoxic through activation of PAR1 and subsequently the phospholipase Cβ-IP3 second messenger system. Inhibition of PAR1 or IP3 prevented granzyme B toxicity. IL-1β enhanced granzyme B-mediated neurotoxicity by increasing PAR1 expression. Conclusions Neurons within the inflamed central nervous system are imperiled because they express more PAR1 and are exposed to a neurotoxic combination of both granzyme B and IL-1β. The effects of these inflammatory mediators may be a contributing factor in the progressive brain atrophy associated with neuroinflammatory diseases. Knowledge of how exposure to IL-1β and granzyme B act synergistically to cause neuronal death yields potential novel neuroprotective treatments for neuroinflammatory diseases

Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

<div><p>Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous <i>in vitro</i> model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine. </p> </div

Generation of neural stem cells from cord blood CD34+ cells.

<p>(A) Non adherent cord blood CD34+ cells (Ctrl) were transduced with Sendai virus constructs containing Oct3/4, Sox2, Klf4 and c-Myc and maintained in neural stem cell medium. Adherent cells were observed on day 2 which proliferated rapidly through day 4 post-infection. (B) More than 95% of the generated cells immunostained for nestin (green) and SOX-2 (red), but were OCT4 negative. Cell nuclei were counterstained with DAPI (blue). The cells were subcultured up to passage 7, when a decline in nestin positive cells was first noticed (C). Spontaneously differentiated neuronal like cells were observed when incubated in neural stem cell medium for over a week without changing medium. These cells developed neural sphere like structures from which βIII-tubulin positive cells with long processes grew out (D). When neural stem cells derived from CD34+ cells were placed in neural differentiation medium for two weeks, both βIII-tubulin (green) neurons and GFAP (red) positive astroglia were seen, along with some cell aggregates (E). Immunostaining for NeuN (green) in the nuclei confirms the presence of relatively mature neurons (F). </p

Inhibition of neurogenesis of induced neural stem cells by inflammatory factor, GrB.

<p>Following treatment with GrB, (A) the total number of induced neural stem cells was determined by using cellquanti-blue assay and (B) the number of proliferating cells was determined using an EdU incorporation assay. Representative photomicrographs show EdU (red) incorporation and DAPI stained nuclei (blue). Results are presented as mean±SEM from at least three independent experiments (n=4 for A and n=3 for B). (C), the effect of GrB on neuronal differentiation was studied using induced neural stem cells derived from adult peripheral CD34+ cells. GrB was used to treat the cells in neuronal differentiation medium for 1 week and βIII-tubulin immunostaining (green) was used to characterize the neurons. GrB treatment resulted in decreased βIII-tubulin positive cells. The morphology of the differentiated neurons in GrB treated group shows that they were more immature, with shorter neurites and less complicated networks, compared to untreated control. </p

3D imaging of contact between neural stem cell (left) and activated T cell (right) by ion abrasion scanning electron microscopy (IA-SEM).

<p>(A, B) Representative, orthogonal 2D images from a 17.57 um x 6.30 um x 8.72 um stack volume show the two cells in direct contact with interlocking cellular membranes in the XZ plane (A) and XY plane (B). The XY plane reveals a localization of Golgi apparatus, vesicles, and mitochondria in the activated T cell near the site of contact. Subcellular organelles are well preserved including nucleus (N), nucleolus (Nc), nuclear pore complexes (Npc), multivesicular bodies (Mvb), lysosome (Ly), mitochondria (M), cytoskeleton (C), intermediate filament (IF), Golgi apparatus (G), vesicles (V), and endoplasmic recticulum (ER). (B, Inset) Expanded view of boxed regions in panel (B) of the interlocking membranes at the cell-cell junction (left) and the T cell mitochondria (right) to illustrate level of detail in images. Scale bars are 100 nm. (C) 3D visualization of the SEM image stack show the activated T cell membrane (blue) engulfing an edge of the neural stem cell membrane (gold). The activated T cell spherical shaped nucleus (ivory), compact mitochondria clusters (pink), and complex membrane protrusions are seen in stark contrast with the neural stem cell flat nucleus (ivory), elongated mitochondria (pink), and smooth cell membrane. (C, Inset) Expanded view of the interior of the T-cell showing the T cell nucleus (navy) is pinched in the center, with the cleft line oriented directly toward the site of contact. Mitochondria (pink) are found clustered along the cleft line.</p

Neurons with action potentials and glial cells differentiated from induced neural stem cells from adult peripheral CD34+ cells.

<p>The induced neural stem cells differentiated into βIII- tubulin positive neurons (green, Ai) in neuronal differentiation or GFAP positive astroglia (red, Aii) in astroglial differentiation medium for 1 week. Oligodendrocyte progenitor differentiation was achieved by incubating the neural stem cells with oligodendrocyte differentiation medium. O4 positive cells (green, Aiii, after 2 weeks) and a few of MBP positive cells (red, Aiv, after four weeks) were detected by immunostaining. Spontaneous neuronal differentiation was also observed in low concentration seeded neural stem cell culture (1000 cells/well in a 24 well plate) after incubation in neural stem cell medium for 1 week (Av). Action potentials were recorded from neurons differentiated for 2 weeks using whole cell patch clamp. Action potentials recorded on two separated cells are shown (Avi). Double staining with either VGLUT1 or VGAT with βIII- tubulin showed most βIII- tubulin positive neurons were either glutamatergic or gabaergic neurons while a few of doparminergic neurons were also detected by TH immunostaining after incubation in neuronal differentiation medium for 2 weeks (B). The induced neural stem cells were passaged for 17 passages and immunostained for nestin as a neural stem cell marker. The cells were also cultured in neural differentiation medium for 2 weeks and immunostained for βIII-tubulin to determine their neuronal differentiation capabilities (C). </p

Inhibition of neurogenesis by activated autologous T cells on induced neural stem cells.

<p>Induced neural stem cells derived from adult peripheral CD34+ cells were cocultured with restive (CT) or activated autologous T cells (AT) for 24 hours. (A) After washing with PBS three times, no adherent T cells were observed in induced neural stem cells co-cultivated with restive T cells (CT-iNSC), while adherent T cells were observed in cultures of induced neural stem cells with activated T cells (AT-iNSC). (B) EdU incorporation assay was used to determine the proliferation of iNSC after 24 hours of co-culture. (C) After 7 days in neuronal differentiation medium, neuronal differentiation was studied by immunostaining for βIII-tubulin. The fluorescence was detected using a plate reader at excitation wavelength 495 nm and emission wavelength 519 nm. AT-iNSC coculture resulted in significantly decreased neuronal differentiation compared to non co-cultured control. Morphologically, AT treatment resulted in fewer neurons, which were more locally aggregated, compared to CT treated groups.</p