Deciphering Brain Metastasis Stem Cell Properties From Colorectal Cancer Highlights Specific Stemness Signature and Shared Molecular Features.

BACKGROUND & AIMS: Brain metastases (BMs) from colorectal cancer (CRC) are associated with significant morbidity and mortality, with chemoresistance and short overall survival. Migrating cancer stem cells with the ability to initiate BM have been described in breast and lung cancers. In this study, we describe the identification and characterization of cancer stem cells in BM from CRC. METHODS: Four brain metastasis stem cell lines from patients with colorectal cancer (BM-SC-CRC1 to BM-SC-CRC4) were obtained by mechanical dissociation of patient's tumors and selection of cancer stem cells by appropriate culture conditions. BM-SC-CRCs were characterized in vitro by clonogenic and limiting-dilution assays, as well as immunofluorescence and Western blot analyses. In ovo, a chicken chorioallantoic membrane (CAM) model and in vivo, xenograft experiments using BALB/c-nude mice were realized. Finally, a whole exome and RNA sequencing analyses were performed. RESULTS: BM-SC-CRC formed metaspheres and contained tumor-initiating cells with self-renewal properties. They expressed stem cell surface markers (CD44v6, CD44, and EpCAM) in serum-free medium and CRC markers (CK19, CK20 and CDX-2) in fetal bovine serum-enriched medium. The CAM model demonstrated their invasive and migratory capabilities. Moreover, mice intracranial xenotransplantation of BM-SC-CRCs adequately recapitulated the original patient BM phenotype. Finally, transcriptomic and genomic approaches showed a significant enrichment of invasiveness and specific stemness signatures and highlighted KMT2C as a potential candidate gene to potentially identify high-risk CRC patients. CONCLUSIONS: This original study represents the first step in CRC BM initiation and progression comprehension, and further investigation could open the way to new therapeutics avenues to improve patient prognosis.

Host-to-graft Propagation of α-synuclein in a Mouse Model of Parkinson's Disease: Intranigral Versus Intrastriatal Transplantation.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and by the accumulation of misfolded α-synuclein (α-syn) in Lewy bodies. Ectopic transplantation of human fetal ventral mesencephalic DA neurons into the striatum of PD patients have provided proof-of-principle for the cell replacement strategy in this disorder. However, 10 to 22 y after transplantation, 1% to 27% of grafted neurons contained α-syn aggregates similar to those observed in the host brain. We hypothesized that intrastriatal grafts are more vulnerable to α-syn propagation because the striatum is not the ontogenic site of nigral DA neurons and represents an unfavorable environment for transplanted neurons. Here, we compared the long-term host-to-graft propagation of α-syn in 2 transplantation sites: the SNpc and the striatum. METHODS: Two mouse models of PD were developed by injecting adeno-associated-virus2/9-human α-syn A53T into either the SNpc or the striatum of C57BL/6 mice. Mouse fetal ventral mesencephalic DA progenitors were grafted into the SNpc or into the striatum of SNpc or striatum of α-syn injected mice, respectively. RESULTS: First, we have shown a degeneration of the nigrostriatal pathway associated with motor deficits after nigral but not striatal adeno-associated-virus-hαsyn A53T injection. Second, human α-syn preferentially accumulates in striatal grafts compared to nigral grafts. However, no differences were observed for phosphorylated α-syn, a marker of pathological α-syn aggregates. CONCLUSIONS: Taken together, our results suggest that the ectopic site of the transplantation impacts the host-to-graft transmission of α-syn.

Beneficial Effects of Hyaluronan-Based Hydrogel Implantation after Cortical Traumatic Injury.

Traumatic brain injury (TBI) causes cell death mainly in the cerebral cortex. We have previously reported that transplantation of embryonic cortical neurons immediately after cortical injury allows the anatomical reconstruction of injured pathways and that a delay between cortical injury and cell transplantation can partially improve transplantation efficiency. Biomaterials supporting repair processes in combination with cell transplantation are in development. Hyaluronic acid (HA) hydrogel has attracted increasing interest in the field of tissue engineering due to its attractive biological properties. However, before combining the cell with the HA hydrogel for transplantation, it is important to know the effects of the implanted hydrogel alone. Here, we investigated the therapeutic effect of HA on host tissue after a cortical trauma. For this, we implanted HA hydrogel into the lesioned motor cortex of adult mice immediately or one week after a lesion. Our results show the vascularization of the implanted hydrogel. At one month after HA implantation, we observed a reduction in the glial scar around the lesion and the presence of the newly generated oligodendrocytes, immature and mature neurons within the hydrogel. Implanted hydrogel provides favorable environments for the survival and maturation of the newly generated neurons. Collectively, these results suggest a beneficial effect of biomaterial after a cortical traumatic injury.

Long-Term Evaluation of Intranigral Transplantation of Human iPSC-Derived Dopamine Neurons in a Parkinson's Disease Mouse Model.

Parkinson's disease (PD) is a neurodegenerative disorder associated with loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). One strategy for treating PD is transplantation of DA neuroblasts. Significant advances have been made in generating midbrain DA neurons from human pluripotent stem cells. Before these cells can be routinely used in clinical trials, extensive preclinical safety studies are required. One of the main issues to be addressed is the long-term therapeutic effectiveness of these cells. In most transplantation studies using human cells, the maturation of DA neurons has been analyzed over a relatively short period not exceeding 6 months. In present study, we generated midbrain DA neurons from human induced pluripotent stem cells (hiPSCs) and grafted these neurons into the SNpc in an animal model of PD. Graft survival and maturation were analyzed from 1 to 12 months post-transplantation (mpt). We observed long-term survival and functionality of the grafted neurons. However, at 12 mpt, we observed a decrease in the proportion of SNpc DA neuron subtype compared with that at 6 mpt. In addition, at 12 mpt, grafts still contained immature neurons. Our results suggest that longer-term evaluation of the maturation of neurons derived from human stem cells is mandatory for the safe application of cell therapy for PD.

Better Outcomes with Intranigral versus Intrastriatal Cell Transplantation: Relevance for Parkinson's Disease.

Intrastriatal embryonic ventral mesencephalon grafts have been shown to integrate, survive, and reinnervate the host striatum in clinical settings and in animal models of Parkinson's disease. However, this ectopic location does not restore the physiological loops of the nigrostriatal pathway and promotes only moderate behavioral benefits. Here, we performed a direct comparison of the potential benefits of intranigral versus intrastriatal grafts in animal models of Parkinson's disease. We report that intranigral grafts promoted better survival of dopaminergic neurons and that only intranigral grafts induced recovery of fine motor skills and normalized cortico-striatal responses. The increase in the number of toxic activated glial cells in host tissue surrounding the intrastriatal graft, as well as within the graft, may be one of the causes of the increased cell death observed in the intrastriatal graft. Homotopic localization of the graft and the subsequent physiological cell rewiring of the basal ganglia may be a key factor in successful and beneficial cell transplantation procedures.

Neuroprotective Effects of Neuropeptide Y against Neurodegenerative Disease.

Neuropeptide Y (NPY), a 36 amino acid peptide, is widely expressed in the mammalian brain. Changes in NPY levels in different brain regions and plasma have been described in several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, and Machado-Joseph disease. The changes in NPY levels may reflect the attempt to set up an endogenous neuroprotective mechanism to counteract the degenerative process. Accumulating evidence indicates that NPY can function as an anti-apoptotic, anti-inflammatory, and pro-phagocytic agent, which may be used effectively to halt or to slow down the progression of the disease. In this review, we will focus on the neuroprotective roles of NPY in several neuropathological conditions, with a particular focus on the anti-inflammatory properties of NPY.

Characterization of Inflammation in Delayed Cortical Transplantation.

We previously reported that embryonic motor cortical neurons transplanted 1-week after lesion in the adult mouse motor cortex significantly enhances graft vascularization, survival, and proliferation of grafted cells, the density of projections developed by grafted neurons and improves functional repair and recovery. The purpose of the present study is to understand the extent to which post-traumatic inflammation following cortical lesion could influence the survival of grafted neurons and the development of their projections to target brain regions and conversely how transplanted cells can modulate host inflammation. For this, embryonic motor cortical tissue was grafted either immediately or with a 1-week delay into the lesioned motor cortex of adult mice. Immunohistochemistry (IHC) analysis was performed to determine the density and cell morphology of resident and peripheral infiltrating immune cells. Then, in situ hybridization (ISH) was performed to analyze the distribution and temporal mRNA expression pattern of pro-inflammatory or anti-inflammatory cytokines following cortical lesion. In parallel, we analyzed the protein expression of both M1- and M2-associated markers to study the M1/M2 balance switch. We have shown that 1-week after the lesion, the number of astrocytes, microglia, oligodendrocytes, and CD45+ cells were significantly increased along with characteristics of M2 microglia phenotype. Interestingly, the majority of microglia co-expressed transforming growth factor-ß1 (TGF-ß1), an anti-inflammatory cytokine, supporting the hypothesis that microglial activation is also neuroprotective. Our results suggest that the modulation of post-traumatic inflammation 1-week after cortical lesion might be implicated in the improvement of graft vascularization, survival, and density of projections developed by grafted neurons.

A Delay between Motor Cortex Lesions and Neuronal Transplantation Enhances Graft Integration and Improves Repair and Recovery.

We previously reported that embryonic motor cortical neurons transplanted immediately after lesions in the adult mouse motor cortex restored damaged motor cortical pathways. A critical barrier hindering the application of transplantation strategies for a wide range of traumatic injuries is the determination of a suitable time window for therapeutic intervention. Here, we report that a 1 week delay between the lesion and transplantation significantly enhances graft vascularization, survival, and proliferation of grafted cells. More importantly, the delay dramatically increases the density of projections developed by grafted neurons and improves functional repair and recovery as assessed by intravital dynamic imaging and behavioral tests. These findings open new avenues in cell transplantation strategies as they indicate successful brain repair may occur following delayed transplantation.SIGNIFICANCE STATEMENT Cell transplantation represents a promising therapy for cortical trauma. We previously reported that embryonic motor cortical neurons transplanted immediately after lesions in the adult mouse motor cortex restored damaged cortical pathways. A critical barrier hindering the application of transplantation strategies for a wide range of traumatic injuries is the determination of a suitable time window for therapeutic intervention. We demonstrate that a 1 week delay between the lesion and transplantation significantly enhances graft vascularization, survival, proliferation, and the density of the projections developed by grafted neurons. More importantly, the delay has a beneficial impact on functional repair and recovery. These results impact the effectiveness of transplantation strategies in a wide range of traumatic injuries for which therapeutic intervention is not immediately feasible.

Collapsin response-mediator protein 5 (CRMP5) phosphorylation at threonine 516 regulates neurite outgrowth inhibition.

The collapsin response-mediator proteins (CRMPs) are multifunctional proteins highly expressed during brain development but down-regulated in the adult brain. They are involved in axon guidance and neurite outgrowth signalling. Among these, the intensively studied CRMP2 has been identified as an important actor in axon outgrowth, this activity being correlated with the reorganisation of cytoskeletal proteins via the phosphorylation state of CRMP2. Another member, CRMP5, restricts the growth-promotional effects of CRMP2 by inhibiting dendrite outgrowth at early developmental stages. This inhibition occurs when CRMP5 binds to tubulin and the microtubule-associated protein MAP2, but the role of CRMP5 phosphorylation is still unknown. Here, we have studied the role of CRMP5 phosphorylation by mutational analysis. Using non-phosphorylatable truncated constructs of CRMP5 we have demonstrated that, among the four previously identified CRMP5 phosphorylation sites (T509, T514, T516 and S534), only the phosphorylation at T516 residue was needed for neurite outgrowth inhibition in PC12 cells and in cultured C57BL/6J mouse hippocampal neurons. Indeed, the expression of the CRMP5 non-phosphorylated form induced a loss of function of CRMP5 and the mutant mimicking the phosphorylated form induced the growth inhibition function seen in wildtype CRMP5. The T516 phosphorylation was achieved by the glycogen synthase kinase-3ß (GSK-3ß), which can phosphorylate the wildtype protein but not the non-phosphorylatable mutant. Furthermore, we have shown that T516 phosphorylation is essential for the tubulin-binding property of CRMP5. Therefore, CRMP5-induced growth inhibition is dependent on T516 phosphorylation through the GSK-3ß pathway. The findings provide new insights into the mechanisms underlying neurite outgrowth.

Alpha-synuclein spreading in M83 mice brain revealed by detection of pathological α-synuclein by enhanced ELISA.

BACKGROUND: The accumulation of misfolded proteins appears as a fundamental pathogenic process in human neurodegenerative diseases. In the case of synucleinopathies such as Parkinson's disease (PD) or dementia with Lewy bodies (DLB), the intraneuronal deposition of aggregated alpha-synuclein (αS) is a major characteristic of the disease, but the molecular basis distinguishing the disease-associated protein (αSD) from its normal counterpart remains poorly understood. However, recent research suggests that a prion-like mechanism could be involved in the inter-cellular and inter-molecular propagation of aggregation of the protein within the nervous system. RESULTS: Our data confirm our previous observations of disease acceleration in a transgenic mouse line (M83) overexpressing a mutated (A53T) form of human αS, following inoculation of either brain extracts from sick M83 mice or fibrillar recombinant αS. A similar phenomenon is observed following a "second passage" in the M83 mouse model, including after stereotactic inoculations into the hippocampus or cerebellum. For further molecular analyses of αSD, we designed an ELISA test that identifies αSD specifically in sick mice and in the brain regions targeted by the pathological process in this mouse model. αSD distribution, mainly in the caudal brain regions and spinal cord, overall appears remarkably uniform, whatever the conditions of experimental challenge. In addition to specific detection of αSD immunoreactivity using an antibody against Ser129 phosphorylated αS, similar results were observed in ELISA with several other antibodies against the C-terminal part of αS, including an antibody against non phosphorylated αS. This also indicated consistent immunoreactivity of the murine αS protein specifically in the affected brain regions of sick mice. CONCLUSIONS: Prion-like behaviour in propagation of the disease-associated αS was confirmed with the M83 transgenic mouse model, that could be followed by an ELISA test. The ELISA data question their possible relationship with the conformational differences between the disease-associated αS and its normal counterpart.

Collapsin response mediator protein 5 (CRMP5) induces mitophagy, thereby regulating mitochondrion numbers in dendrites.

Degradation of damaged mitochondria by mitophagy is an essential process to ensure cell homeostasis. Because neurons, which have a high energy demand, are particularly dependent on the mitochondrial dynamics, mitophagy represents a key mechanism to ensure correct neuronal function. Collapsin response mediator proteins 5 (CRMP5) belongs to a family of cytosolic proteins involved in axon guidance and neurite outgrowth signaling during neural development. CRMP5, which is highly expressed during brain development, plays an important role in the regulation of neuronal polarity by inhibiting dendrite outgrowth at early developmental stages. Here, we demonstrated that CRMP5 was present in vivo in brain mitochondria and is targeted to the inner mitochondrial membrane. The mitochondrial localization of CRMP5 induced mitophagy. CRMP5 overexpression triggered a drastic change in mitochondrial morphology, increased the number of lysosomes and double membrane vesicles termed autophagosomes, and enhanced the occurrence of microtubule-associated protein 1 light chain 3 (LC3) at the mitochondrial level. Moreover, the lipidated form of LC3, LC3-II, which triggers autophagy by insertion into autophagosomes, enhanced mitophagy initiation. Lysosomal marker translocates at the mitochondrial level, suggesting autophagosome-lysosome fusion, and induced the reduction of mitochondrial content via lysosomal degradation. We show that during early developmental stages the strong expression of endogenous CRMP5, which inhibits dendrite growth, correlated with a decrease of mitochondrial content. In contrast, the knockdown or a decrease of CRMP5 expression at later stages enhanced mitochondrion numbers in cultured neurons, suggesting that CRMP5 modulated these numbers. Our study elucidates a novel regulatory mechanism that utilizes CRMP5-induced mitophagy to orchestrate proper dendrite outgrowth and neuronal function.

Identification of a new CRMP5 isoform present in the nucleus of cancer cells and enhancing their proliferation.

Collapsin Response Mediator Protein 5 (CRMP5) belongs to a family of five cytosolic proteins highly expressed in the developing nervous system but downregulated in the adult brain. When expressed at the adult stage, CRMP5 is involved in neurological disorders. Indeed, CRMP5 is found expressed in cancer cells of some brain tumors, such as glioblastoma, or in small cell lung cancer causing paraneoplastic neurological syndromes as a result of cancer-induced auto-immune processes. Nevertheless, its role in cancer pathology is still obscure. Here, we show a new short isoform, derived from C-terminal processing of CRMP5, presenting a nuclear localization both in human glioblastoma, and in cancer cell lines (H69, GL15). By mutational analysis, we demonstrate that nuclear translocation occurs via nuclear localization signal (NLS), where the essential residue for nuclear location is K391. Direct CRMP5/ tubulin interaction, previously shown during brain development, does not occur for cytosolic CRMP5 in pathological conditions, leading to the suggestion that in cancer cells CRMP5 is not sequestered in the cytosol; therefore it may undergo C-terminal truncation allowing the exposure of the NLS for active translocation. Moreover, we show that the function associated with the CRMP5 nuclear targeting is an increase of cell proliferation activity.

CRMP5 interacts with tubulin to inhibit neurite outgrowth, thereby modulating the function of CRMP2.

Collapsin response mediator proteins (CRMPs) are involved in signaling of axon guidance and neurite outgrowth during neural development and regeneration. Among these, CRMP2 has been identified as an important actor in neuronal polarity and axon outgrowth, these activities being correlated with the reorganization of cytoskeletal proteins. In contrast, the function of CRMP5, expressed during brain development, remains obscure. Here, we find that, in contrast to CRMP2, CRMP5 inhibits tubulin polymerization and neurite outgrowth. Knockdown of CRMP5 expression by small interfering RNA confirms its inhibitory functions. CRMP5 forms a ternary complex with MAP2 and tubulin, the latter involving residues 475-522 of CRMP5, exposed at the molecule surface. Using different truncated CRMP5 constructs, we demonstrate that inhibition of neurite outgrowth by CRMP5 is mediated by tubulin binding. When both CRMP5 and CRMP2 are overexpressed, the inhibitory effect of CRMP5 abrogates neurite outgrowth promotion induced by CRMP2, suggesting that CRMP5 acts as a dominant signal. In cultured hippocampal neurons, CRMP5 shows no effect on axon growth, whereas it inhibits dendrite outgrowth and formation, at an early developmental stage, correlated with its strong expression in neurites. At later stages, when dendrites begin to extend, CRMP5 expression is absent. However, CRMP2 is constantly expressed. Overexpression of CRMP5 with CRMP2 inhibits CRMP2-induced outgrowth both on the axonal and dendritic levels. Deficiency of CRMP5 expression enhanced the CRMP2 effect. This antagonizing effect of CRMP5 is exerted through a tubulin-based mechanism. Thus, the CRMP5 binding to tubulin modulates CRMP2 regulation of neurite outgrowth and neuronal polarity during brain development.