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.

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...

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-) 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.

Background: Apolipoprotein E4 (apoE4), the major gene involved in Alzheimer disease, has a unique structure, intramolecular domain interaction, that is associated with neuropathology. Results: Potent small molecule structure correctors block apoE4 domain interaction and reverse apoE4 detrimental effects in cultured neurons. Conclusion: Structure correctors negate the detrimental effects of apoE4 in neurons. Significance: ApoE4 structure correctors could represent a therapeutic approach for treating apoE4-associated neuropathology.

Neuronal expression of apolipoprotein (apo) E4 may contribute to the pathogenesis of Alzheimer's disease (AD). In studying how apoE expression is regulated in neurons, we identified a splicing variant of apoE mRNA with intron-3 retention (apoE-I3). ApoE-I3 mRNA was detected in neuronal cell lines and primary neurons, but not in astrocytic cell lines or primary astrocytes, from humans and mice by reverse transcription (RT)-PCR. In both wild-type and human apoE knock-in mice, apoE-I3 was found predominantly in cortical and hippocampal neurons by in situ hybridization. Cell fractionation and quantitative RT-PCR revealed that over 98% of the apoE-I3 mRNA was retained in the nucleus without protein translation. In transfected primary neurons, apoE expression increased dramatically when intron-3 was deleted from a genomic DNA construct and decreased markedly when intron-3 was inserted into a cDNA construct, suggesting that intron-3 retention/splicing controls apoE expression in neurons. In response to excitotoxic challenge, the apoE-I3 mRNA was markedly increased in morphologically normal hippocampal neurons but reduced in degenerating hippocampal neurons in mice; apoE mRNA showed the opposite pattern. This apparent precursor-product relationship between apoE-I3 and apoE mRNA was supported by a transcriptional inhibition study. Thus, neuronal expression of apoE is controlled by transcription of apoE-I3 under normal conditions and by processing of apoE-I3 into mature apoE mRNA in response to injury.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.The three isoforms of human apoE (apoE2, apoE3, and apoE4) differ at amino acid positions 112 or 158 or both (1, 2). ApoE4 is the major genetic risk factor for Alzheimer disease (AD) 2 (3-5), and apoE4 carriers account for 65-80% of all AD cases, highlighting the importance of apoE4 in AD pathogenesis (6). Two biophysical properties that distinguish apoE4 from the other isoforms likely hold the key to a mechanistic understanding of its association with AD. First, apoE4 is more unstable and tends to form a molten globule state (7,8). Second, the amino-terminal domain (amino acids 1-191) of apoE4 interacts with its carboxyl-terminal domain (amino acids 223-299) (9, 10). This domain interaction occurs predominantly in apoE4, in which positively charged Arg-112 repels the side chain of Arg-61 in the amino-terminal domain, allowing the formation of a salt bridge between Arg-61 and the negatively charged Glu-255 in the carboxyl-terminal domain (9, 10). Domain interaction occurs to a significantly lesser extent in apoE2 and apoE3 because both have Cys-112, resulting in a different conformation of Arg-61 (11). Importantly, only human apoE has Arg-61; the 17 other species in which the apoE gene has been sequenced have 11). Mutation of Arg-61 to Thr in apoE4 prevents domain interaction, converting apoE4 to an apoE3-like molecule (9 -12). ApoE4 domain interaction occurs on lipoprotein particles in vitro in human plasma, in cultured Neuro-2a cells, and in Arg-61 knock-in mice, in wh...

SummaryTauopathies represent a group of neurodegenerative disorders characterized by the accumulation of pathological TAU protein in brains. We report a human neuronal model of tauopathy derived from induced pluripotent stem cells (iPSCs) carrying a TAU-A152T mutation. Using zinc-finger nuclease-mediated gene editing, we generated two isogenic iPSC lines: one with the mutation corrected, and another with the homozygous mutation engineered. The A152T mutation increased TAU fragmentation and phosphorylation, leading to neurodegeneration and especially axonal degeneration. These cellular phenotypes were consistent with those observed in a patient with TAU-A152T. Upon mutation correction, normal neuronal and axonal morphologies were restored, accompanied by decreases in TAU fragmentation and phosphorylation, whereas the severity of tauopathy was intensified in neurons with the homozygous mutation. These isogenic TAU-iPSC lines represent a critical advancement toward the accurate modeling and mechanistic study of tauopathies with human neurons and will be invaluable for drug-screening efforts and future cell-based therapies.