No abstract

Accumulation of misfolded proteins and alterations in Ca2؉ homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death. However, the signal-transducing events that connect ER stress to cell death pathways are incompletely understood. To discern the pathway by which ER stress-induced cell death proceeds, we performed studies on Apaf-1 ؊/؊ (null) fibroblasts that are known to be relatively resistant to apoptotic insults that induce the intrinsic apoptotic pathway. While these cells were resistant to cell death initiated by proapoptotic stimuli such as tamoxifen, they were susceptible to apoptosis induced by thapsigargin and brefeldin-A, both of which induce ER stress. This pathway was inhibited by catalytic mutants of caspase-12 and caspase-9 and by a peptide inhibitor of caspase-9 but not by caspase-8 inhibitors. Cleavage of caspases and poly(ADP-ribose) polymerase was observed in cell-free extracts lacking cytochrome c that were isolated from thapsigargin or brefeldin-treated cells. To define the molecular requirements for this Apaf-1 and cytochrome c-independent apoptosis pathway further, we developed a cell-free system of ER stress-induced apoptosis; the addition of microsomes prepared from ER stress-induced cells to a normal cell extract lacking mitochondria or cytochrome c resulted in processing of caspases. Immunodepletion experiments suggested that caspase-12 was one of the microsomal components required to activate downstream caspases. Thus, ER stress-induced programmed cell death defines a novel, mitochondrial and Apaf-1-independent, intrinsic apoptotic pathway.

Macroautophagy (hereafter autophagy) is a key pathway in neurodegeneration. Despite protective actions, autophagy may contribute to neuron demise, when dysregulated. Here we considered 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 considered 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 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.

In vitro, the overexpression of the bcl-2 protooncogene in cultured neurons has been shown to prevent apoptosis induced by neurotrophic factor deprivation. We have generated transgenic mice overexpressing the Bc1-2 protein in neurons, including motoneurons of the facial nucleus. We have tested whether Bcl-2 could protect these motoneurons from experimentally induced cell death in new born mice. To address this question, we performed unilateral lesion of the facial nerve of wild-type and transgenic 2-day-old mice. In wild-type mice, the lesioned nerve and the corresponding motoneuron cell bodies in the facial nucleus underwent rapid degeneration. In contrast, in transgenic mice, facial motoneurons survived axotomy. Not only their cell bodies but also their axons were protected up to the lesion site. These results demonstrate that in vivo Bcl-2 protects neonatal motoneurons from degeneration after axonal injury. A better understanding of the mechanisms by which Bc1-2 prevents neuronal cell death in vivo could lead to the development of strategies for the treatment of motoneuron degenerative diseases.Apoptotic neuronal cell death that occurs during development of the nervous system requires protein synthesis (1,2) and is regulated by epigenetic factors such as retrograde neurotrophic factors (3,4). Apoptosis probably results from an unbalance between positive and negative regulators of cell survival (5). One positive regulator identified in vertebrates is the Bcl-2 oncoprotein (6)(7)(8). In vitro, this 25-kDa membrane-associated protein (9)(10)(11) is capable of rescuing neurons from apoptosis induced by neurotrophic factor deprivation (12)(13)(14). These observations led to the hope that Bcl-2 could be a tool for the treatment of neurodegenerative diseases although there was no evidence that the protein could block neuronal death under pathological circumstances in vivo. One ofthe reasons to challenge this hope was that the type of cell death occurring in vivo (15) could be different from classical apoptosis described in vitro (2, 16). We therefore decided to test the effects of Bcl-2 in vivo, on transgenic mice, in a model of nerve injury that leads to neuronal cell death. In rodents, during the early postnatal period, lesion of the facial or sciatic nerve leads to a rapid degeneration of the axotomized motoneurons (17)(18)(19). This model has been exploited to investigate the effect of neurotrophic factors on motoneuron survival (20)(21)(22)(23). To determine whether overexpression of Bcl-2 protects axotomized motoneurons, we generated transgenic mice in which neurons overexpress Bcl-2. Unilateral section ofthe facial nerve was performed on 2-day-old transgenic mice, in which facial motoneurons overexpress the Bcl-2 protein, and on wild-type pups from the same litter. Seven days after the lesion, motoneuron cell bodies were always present in the ipsilateral facial nucleus in transgenic mice but had degenerated and disappeared from the ipsilateral facial nucleus in the wild-type animals. MATERIALS AND MET...

SummaryDeveloping 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.