Jessica E. Young

SOMATIC CODING MUTATIONS IN HUMAN INDUCED PLURIPOTENT STEM CELLS

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.

Nutrient deprivation induces neuronal autophagy and implicates reduced insulin signaling in neuroprotective autophagy activation.

Although autophagy maintains normal neural function by degrading misfolded proteins, little is known about how neurons activate this integral response. Furthermore, classical methods of autophagy induction used with nonneural cells, such as starvation, simply result in neuron death. To study neuronal autophagy, we cultured primary cortical neurons from transgenic mice that ubiquitously express green fluorescent protein-tagged LC3 and monitored LC3-I to LC3-II conversion by immunohistochemistry and immunoblotting. Evaluation of different culture media led us to discover that culturing primary neurons in Dulbeccos modified Eagles medium without B27 supplementation robustly activates autophagy. We validated this nutrient-limited media approach for inducing autophagy by showing that 3-methyl-adenine treatment and Atg5 RNA interference knockdown each inhibits LC3-I to LC3-II conversion. Evaluation of B27 supplement components yielded insulin as the factor whose absence induced autophagy in primary neurons, and this activation was mammalian target of rapamycin-dependent. When we tested if nutrient-limited media could protect neurons expressing polyglutamine-expanded proteins against cell death, we observed a strong protective effect, probably due to autophagy activation. Our results indicate that nutrient deprivation can be used to understand the regulatory basis of neuronal autophagy and implicate diminished insulin signaling in the activation of neuronal autophagy.

The Presenilin-1 ΔE9 Mutation Results in Reduced γ-Secretase Activity, but Not Total Loss of PS1 Function, in Isogenic Human Stem Cells

Presenilin 1 (PS1) is the catalytic core of γ-secretase, which cleaves type 1 transmembrane proteins, including the amyloid precursor protein (APP). PS1 also has γ-secretase-independent functions, and dominant PS1 missense mutations are the most common cause of familial Alzheimers disease (FAD). Whether PS1 FAD mutations are gain- or loss-of-function remains controversial, primarily because most studies have relied on overexpression in mouse and/or nonneuronal systems. We used isogenic euploid human induced pluripotent stem cell lines to generate and study an allelic series of PS1 mutations, including heterozygous null mutations and homozygous and heterozygous FAD PS1 mutations. Rigorous analysis of this allelic series in differentiated, purified neurons allowed us to resolve this controversy and to conclude that FAD PS1 mutations, expressed at normal levels in the appropriate cell type, impair γ-secretase activity but do not disrupt γ-secretase-independent functions of PS1. Thus, FAD PS1 mutations do not act as simple loss of PS1 function but instead dominantly gain an activity toxic to some, but not all, PS1 functions.

Polyglutamine-expanded androgen receptor interferes with TFEB to elicit autophagy defects in SBMA

Macroautophagy (hereafter autophagy) is a key pathway in neurodegeneration. Despite protective actions, autophagy may contribute to neuron demise when dysregulated. Here we consider X-linked spinal and bulbar muscular atrophy (SBMA), a repeat disorder caused by polyglutamine-expanded androgen receptor (polyQ-AR). We found that polyQ-AR reduced long-term protein turnover and impaired autophagic flux in motor neuron–like cells. Ultrastructural analysis of SBMA mice revealed a block in autophagy pathway progression. We examined the transcriptional regulation of autophagy and observed a functionally significant physical interaction between transcription factor EB (TFEB) and AR. Normal AR promoted, but polyQ-AR interfered with, TFEB transactivation. To evaluate physiological relevance, we reprogrammed patient fibroblasts to induced pluripotent stem cells and then to neuronal precursor cells (NPCs). We compared multiple SBMA NPC lines and documented the metabolic and autophagic flux defects that could be rescued by TFEB. Our results indicate that polyQ-AR diminishes TFEB function to impair autophagy and promote SBMA pathogenesis.

Elucidating Molecular Phenotypes Caused by the SORL1 Alzheimer’s Disease Genetic Risk Factor Using Human Induced Pluripotent Stem Cells

Predisposition to sporadic Alzheimers disease (SAD) involves interactions between a persons unique combination of genetic variants and the environment. The molecular effect of these variants may be subtle and difficult to analyze with standard in vitro or in vivo models. Here we used hIPSCs to examine genetic variation in the SORL1 gene and possible contributions to SAD-related phenotypes in human neurons. We found that human neurons carrying SORL1 variants associated with an increased SAD risk show a reduced response to treatment with BDNF, at the level of both SORL1 expression and APP processing. shRNA knockdown of SORL1 demonstrates that the differences in BDNF-induced APP processing between genotypes are dependent on SORL1 expression. We propose that the variation in SORL1 expression induction by BDNF is modulated by common genetic variants and can explain how genetic variation in this one locus can contribute to an individuals risk of developing SAD.

Proteolytic Cleavage of Ataxin-7 by Caspase-7 Modulates Cellular Toxicity and Transcriptional Dysregulation

Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by specific degeneration of cerebellar, brainstem, and retinal neurons. Although they share little sequence homology, proteins implicated in polyQ disorders have common properties beyond their characteristic polyQ tract. These include the production of proteolytic fragments, nuclear accumulation, and processing by caspases. Here we report that ataxin-7 is cleaved by caspase-7, and we map two putative caspase-7 cleavage sites to Asp residues at positions 266 and 344 of the ataxin-7 protein. Site-directed mutagenesis of these two caspase-7 cleavage sites in the polyQ-expanded form of ataxin-7 produces an ataxin-7 D266N/D344N protein that is resistant to caspase cleavage. Although ataxin-7 displays toxicity, forms nuclear aggregates, and represses transcription in human embryonic kidney 293T cells in a polyQ length-dependent manner, expression of the non-cleavable D266N/D344N form of polyQ-expanded ataxin-7 attenuated cell death, aggregate formation, and transcriptional interference. Expression of the caspase-7 truncation product of ataxin-7-69Q or -92Q, which removes the putative nuclear export signal and nuclear localization signals of ataxin-7, showed increased cellular toxicity. We also detected N-terminal polyQ-expanded ataxin-7 cleavage products in SCA7 transgenic mice similar in size to those generated by caspase-7 cleavage. In a SCA7 transgenic mouse model, recruitment of caspase-7 into the nucleus by polyQ-expanded ataxin-7 correlated with its activation. Our results, thus, suggest that proteolytic processing of ataxin-7 by caspase-7 may contribute to SCA7 disease pathogenesis.

Polyglutamine-Expanded Androgen Receptor Truncation Fragments Activate a Bax-Dependent Apoptotic Cascade Mediated by DP5/Hrk

Spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder caused by a polyglutamine (polyQ) repeat expansion in the androgen receptor (AR). PolyQ-AR neurotoxicity may involve generation of an N-terminal truncation fragment, as such peptides occur in SBMA patients and mouse models. To elucidate the basis of SBMA, we expressed N-terminal truncated AR in motor neuron-derived cells and primary cortical neurons. Accumulation of polyQ-AR truncation fragments in the cytosol resulted in neurodegeneration and apoptotic, caspase-dependent cell death. Using primary neurons from mice transgenic or deficient for apoptosis-related genes, we determined that polyQ-AR apoptotic activation is fully dependent on Bax. Jun N-terminal kinase (JNK) was required for apoptotic pathway activation through phosphorylation of c-Jun. Expression of polyQ-AR in DP5/Hrk null neurons yielded significant protection against apoptotic activation, but absence of Bim did not provide protection, apparently due to compensatory upregulation of DP5/Hrk or other BH3-only proteins. Misfolded AR protein in the cytosol thus initiates a cascade of events beginning with JNK and culminating in Bax-dependent, intrinsic pathway activation, mediated in part by DP5/Hrk. As apoptotic mediators are candidates for toxic fragment generation and other cellular processes linked to neuron dysfunction, delineation of the apoptotic activation pathway induced by polyQ-expanded AR may shed light on the pathogenic cascade in SBMA and other motor neuron diseases.

Defective Transcytosis of APP and Lipoproteins in Human iPSC-Derived Neurons with Familial Alzheimer’s Disease Mutations

We investigated early phenotypes caused by familial Alzheimers disease (fAD) mutations in isogenic human iPSC-derived neurons. Analysis of neurons carrying fAD PS1 or APP mutations introduced using genome editing technology at the endogenous loci revealed that fAD mutant neurons had previously unreported defects in the recycling state of endocytosis and soma-to-axon transcytosis of APP and lipoproteins. The endocytosis reduction could be rescued through treatment with a β-secretase inhibitor. Our data suggest that accumulation of β-CTFs of APP, but not Aβ, slow vesicle formation from an endocytic recycling compartment marked by the transcytotic GTPase Rab11. We confirm previous results that endocytosis is affected in AD and extend these to uncover a neuron-specific defect. Decreased lipoprotein endocytosis and transcytosis to the axon suggest that a neuron-specific impairment in endocytic axonal delivery of lipoproteins and other key materials might compromise synaptic maintenance in fAD.

Development of selective nutrient deprivation as a system to study autophagy induction and regulation in neurons

Autophagy is emerging as a fundamentally important pathway for countering misfolded protein stress in the central nervous system. Indeed, many studies suggest that upregulation of a properly functioning macroautophagy pathway can be neuroprotective in neurodegenerative disorders characterized by the production of toxic protein conformers. Despite these advances, little is known about how autophagy is regulated in neurons. To directly study neuronal autophagy, we developed a primary neuron culture system where we can induce autophagy by withdrawal of a key supplement from the culture medium. We recently reported that the absence of insulin from the culture medium induces autophagy in this primary neuron system, and that the neuronal autophagy activation is mTOR-dependent. Further studies indicate that our nutrient-deprivation method of autophagy induction yields normally functioning and fully progressing autophagy based upon treatment with lysosomal inhibitors. As this method of autophagy induction can protect neurons from proteotoxic cell death, our findings suggest that an understanding of how to turn on autophagy in neurons could translate into a viable approach for treating neurodegenerative proteinopathies. However, before therapeutic applications can be realized, the pathways regulating neuronal autophagy need to be defined. As highlighted herein, our system for autophagy induction should contribute to efforts aimed at understanding the regulatory basis of autophagy activation in neurons.

Stabilizing the Retromer Complex in a Human Stem Cell Model of Alzheimer’s Disease Reduces TAU Phosphorylation Independently of Amyloid Precursor Protein

Summary Developing effective therapeutics for complex diseases such as late-onset, sporadic Alzheimer’s disease (SAD) is difficult due to genetic and environmental heterogeneity in the human population and the limitations of existing animal models. Here, we used hiPSC-derived neurons to test a compound that stabilizes the retromer, a highly conserved multiprotein assembly that plays a pivotal role in trafficking molecules through the endosomal network. Using this human-specific system, we have confirmed previous data generated in murine models and show that retromer stabilization has a potentially beneficial effect on amyloid beta generation from human stem cell-derived neurons. We further demonstrate that manipulation of retromer complex levels within neurons affects pathogenic TAU phosphorylation in an amyloid-independent manner. Taken together, our work demonstrates that retromer stabilization is a promising candidate for therapeutic development in AD and highlights the advantages of testing novel compounds in a human-specific, neuronal system.