Yuki Nakamura

Homologues of the Musashi family of RNA-binding proteins are evolutionarily conserved across species. In mammals, two members of this family, Musashi1 (Msi1) and Musashi2 (Msi2), are strongly coexpressed in neural precursor cells, including CNS stem cells. To address the in vivo roles of msi in neural development, we generated mice with a targeted disruption of the gene encoding Msi1. Homozygous newborn mice frequently developed obstructive hydrocephalus with aberrant proliferation of ependymal cells in a restricted area surrounding the Sylvius aqueduct. These observations indicate a vital role for msi1 in the normal development of this subpopulation of ependymal cells, which has been speculated to be a source of postnatal CNS stem cells. On the other hand, histological examination and an in vitro neurosphere assay showed that neither the embryonic CNS development nor the self-renewal activity of CNS stem cells in embryonic forebrains appeared to be affected by the disruption of msi1, but the diversity of the cell types produced by the stem cells was moderately reduced by the msi1 deficiency. Therefore, we performed antisense ablation experiments to target both msi1 and msi2 in embryonic neural precursor cells. Administration of the antisense peptide-nucleotides, which were designed to specifically down-regulate msi2 expression, to msi1(-/-) CNS stem cell cultures drastically suppressed the formation of neurospheres in a dose-dependent manner. Antisense-treated msi1(-/-) CNS stem cells showed a reduced proliferative activity. These data suggest that msi1 and msi2 are cooperatively involved in the proliferation and maintenance of CNS stem cell populations.

Hes1 is one of the basic helix-loop-helix transcription factors that regulate mammalian CNS development, and its loss- and gain-of-function phenotypes indicate that it negatively regulates neuronal differentiation. Here we report that Hes1(-/-) mice expressed both early (TuJ1 and Hu) and late (MAP2 and Neurofilament) neuronal markers prematurely, and that there were approximately twice the normal number of neurons in the Hes1(-/-) brain during early neural development. However, immunochemical analyses of sections and dissociated cells using neural progenitor markers, including nestin, failed to detect any changes in Hes1(-/-) progenitor population. Therefore, further characterization of neural progenitor cells that discriminated between multipotent and monopotent cells was performed using two culture methods, low-density culture, and a neurosphere assay. We demonstrate that the self-renewal activity of multipotent progenitor cells was reduced in the Hes1(-/-) brain, and that their subsequent commitment to the neuronal lineage was accelerated. The Hes1(-/-) neuronal progenitor cells were functionally abnormal, in that they divided, on average, only once, and then generated two neurons, (instead of one progenitor cell and one neuron), whereas wild-type progenitor cells divided more. In addition, some Hes1(-/-) progenitors followed an apoptotic fate. The overproduction of neurons in the early Hes1(-/-) brains may reflect this premature and immediate generation of neurons as well as a net increase in the number of neuronal progenitor cells. Taken together, we conclude that Hes1 is important for maintaining the self-renewing ability of progenitors and for repressing the commitment of multipotent progenitor cells to a neuronal fate, which is critical for the correct number of neurons to be produced and for the establishment of normal neuronal function.

Musashi1 (Msi1) is a mammalian neural RNA-binding protein highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic and postnatal CNS development. Here, we identified Musashi2 (Msi2), a novel mammalian RNA-binding protein that exhibits high sequence similarity to Msi1. The Msi2 transcript appeared to be distributed ubiquitously in a wide variety of tissues, consistent with the mRNA distribution of its Xenopus homolog, xrp1. However, the present study revealed cell type-specific and developmentally regulated expression of Msi2 in the mammalian CNS. Interestingly, Msi2 was expressed prominently in precursor cells in the ventricular zone and subventricular zone with the same pattern as Msi1 throughout CNS development. In the postnatal and adult CNS, this concurrent expression of Msi2 and Msi1 was seen in cells of the astrocyte lineage, including ependymal cells, a possible source for postnatal CNS stem cells. During neurogenesis, the expression of both Msi2 and Msi1 was lost in most postmitotic neurons, whereas Msi2 expression persisted in a subset of neuronal lineage cells, such as parvalbumin-containing GABA neurons in the neocortex and neurons in several nuclei of the basal ganglia. Msi2 may have a unique role that is required for the generation and/or maintenance of specific neuronal lineages. Furthermore, in vitro studies showed that Msi2 and Msi1 have similar RNA-binding specificity. These two RNA-binding proteins may exert common functions in neural precursor cells by regulating gene expression at the post-transcriptional level.

Cancer cells in the tumour microenvironment use various mechanisms to evade the immune system, particularly T cell attack1. For example, metabolic reprogramming in the tumour microenvironment and mitochondrial dysfunction in tumour-infiltrating lymphocytes (TILs) impair antitumour immune responses2–4. However, detailed mechanisms of such processes remain unclear. Here we analyse clinical specimens and identify mitochondrial DNA (mtDNA) mutations in TILs that are shared with cancer cells. Moreover, mitochondria with mtDNA mutations from cancer cells are able to transfer to TILs. Typically, mitochondria in TILs readily undergo mitophagy through reactive oxygen species. However, mitochondria transferred from cancer cells do not undergo mitophagy, which we find is due to mitophagy-inhibitory molecules. These molecules attach to mitochondria and together are transferred to TILs, which results in homoplasmic replacement. T cells that acquire mtDNA mutations from cancer cells exhibit metabolic abnormalities and senescence, with defects in effector functions and memory formation. This in turn leads to impaired antitumour immunity both in vitro and in vivo. Accordingly, the presence of an mtDNA mutation in tumour tissue is a poor prognostic factor for immune checkpoint inhibitors in patients with melanoma or non-small-cell lung cancer. These findings reveal a previously unknown mechanism of cancer immune evasion through mitochondrial transfer and can contribute to the development of future cancer immunotherapies. Mitochondria with mutations in their DNA from cancer cells can be transferred to T cells in the tumour microenvironment, which leads to T cell dysfunction and impaired antitumour immunity.