SUMMARY The generation of induced pluripotent stem (iPS) cells and induced neuronal (iN) cells from somatic cells provides new avenues for basic research and potential transplantation therapies for neurological diseases. However, clinical applications must consider the risk of tumor formation by iPS cells and the inability of iN cells to self-renew in culture. Here we report the generation of induced neural stem cells (iNSCs) from mouse and human fibroblasts by direct reprogramming with a single factor, Sox2. iNSCs express NSC markers and resemble wild-type NSCs in their morphology, self-renewal, ability to form neurospheres, and gene expression profiles. Cloned iNSCs differentiate into several types of mature neurons, as well as astrocytes and oligodendrocytes, indicating multipotency. Implanted iNSCs can survive and integrate in mouse brains and, unlike iPS cell-derived NSCs, do not generate tumors. Thus, self-renewable and multipotent iNSCs without tumorigenic potential can be generated directly from fibroblasts by reprogramming.

Efforts to develop drugs for Alzheimer’s disease (AD) have shown promise in animal studies, only to fail in human trials, suggesting a pressing need to study AD in human model systems. Using human neurons derived from induced pluripotent stem cells carrying the major genetic risk factor apolipoprotein E4 (apoE4), we demonstrate that apoE4 neurons have higher levels of tau phosphorylation unrelated to their increased Aβ production and displayed GABAergic neuron degeneration. ApoE4 increased Aβ production in human, but not in mouse, neurons. Converting apoE4 to apoE3 by gene editing rescued these phenotypes, indicating the specific effects of apoE4. Neurons lacking apoE behaved like those expressing apoE3, and introducing apoE4 expression recapitulated the pathological phenotypes, suggesting a gain of toxic effects from apoE4. Treating apoE4 neurons with a small-molecule structure corrector ameliorated the detrimental effects, providing a proof of concept that correcting the pathogenic conformation of apoE4 is a viable therapeutic approach for apoE4-related AD.

Apolipoprotein (apo) E4, a 299-aa protein and a major risk factor for Alzheimer's disease, can be cleaved to generate C-terminaltruncated fragments that cause neurotoxicity in vitro and neurodegeneration and behavioral deficits in transgenic mice. To investigate this neurotoxicity, we expressed apoE4 with C-or N-terminal truncations or mutations in transfected Neuro-2a cells. ApoE4 Alzheimer's disease ͉ mitochondria ͉ proteolysis H uman apolipoprotein (apo) E, a 34-kDa protein with 299 aa, has three major isoforms, apoE2, apoE3, and apoE4 (1-4). ApoE4 is a major risk factor for Alzheimer's disease (AD) (5-7). The apoE4 allele, which is found in 40-65% of cases of sporadic and familial AD, increases the occurrence and lowers the age of onset of the disease (7,8).Biochemical, cell biological, transgenic animal, and human studies have suggested several potential mechanisms to explain the contribution of apoE4 to the pathogenesis of AD. These mechanisms include modulation of the deposition and clearance of amyloid ␤ (A␤) peptides and the formation of plaques (9 -15), modulation of A␤-caused synaptic and cholinergic deficits (16), acceleration of age-and excitotoxicity-related neurodegeneration (17), impairment of the antioxidative defense system and mitochondrial function (18 -21), dysregulation of neuronal signaling pathways (22), altered phosphorylation of tau and neurofibrillary tangle formation (23-28), depletion of cytosolic androgen receptor levels in the brain (29, 30), potentiation of A␤-induced lysosomal leakage and apoptosis in neuronal cells (31), and promotion of endosomal abnormalities linked to A␤ overproduction (32-34). The mechanisms of these apoE4-mediated detrimental effects are largely unknown.We have shown that apoE can be cleaved by a neuronspecific chymotrypsin-like serine protease that generates bioactive C-terminal-truncated forms of apoE (25,27,28). The fragments are found at higher levels in the brains of AD patients than in age-and sex-matched controls (27), and apoE4 is more susceptible to cleavage than apoE3. When expressed in cultured neuronal cells or added exogenously to the cultures, apoE4 fragments are neurotoxic, leading to cell death (25). When expressed in transgenic mice, they cause AD-like neurodegeneration and behavioral deficits (27). Because apoE is synthesized by neurons under diverse pathophysiological conditions (35-49), we hypothesize that apoE4 produced in neurons in response to stress or injury (e.g., A␤ toxicity, brain trauma, or oxidative stress) is uniquely susceptible to proteolytic cleavage and that the resulting bioactive C-terminaltruncated fragments induce neuropathology and associated behavioral deficits. ApoE3 also undergoes proteolytic cleavage but to a lesser extent.In this study, we investigated the cellular and molecular mechanisms of the neurotoxicity caused by apoE4 fragments in cultured neuronal cells. We also evaluated the roles of various regions [specifically, the receptor-binding region (amino acids 135-150) and the lipid-binding region (amino acid...

Apolipoprotein (apo-) B mRNA editing is the deamination of cytidine that creates a new termination codon and produces a truncated version of apo-B (apo-B48). The cytidine deaminase catalytic subunit [apo-B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1)] of the multiprotein editing complex has been identified. We generated transgenic rabbits and mice expressing rabbit APOBEC-1 in their livers to determine whether hepatic expression would lower low density lipoprotein cholesterol concentrations. The apo-B mRNA from the livers of the transgenic mice and rabbit was extensively edited, and the transgenic animals had reduced concentrations of apo-B100 and low density lipoproteins compared with control animals. Unexpectedly, all of the transgenic mice and a transgenic rabbit had liver dysplasia, and many transgenic mice developed hepatocellular carcinomas. Many of the mouse livers were hyperplastic and filled with lipid. Other hepatic mRNAs with sequence motifs similar to apo-B mRNA were examined for this type of editing (i.e., cytidine deamination). One of these, tyrosine kinase, was edited in livers of transgenic mice but not of controls. This result demonstrates that other mRNAs can be edited by the overexpressed editing enzyme and suggests that aberrant editing of hepatic mRNAs involved in cell growth and regulation is the cause of the tumorigenesis. Finally, these findings compromise the potential use of APOBEC-1 for gene therapy to lower plasma levels of low density lipoproteins.