Highlights d MLKL expression is induced in the Schwann cells of sciatic nerves following injury d MLKL is phosphorylated at serine 441 site in the Schwann cells of injured nerves d The serine 441 phospho-mimic MLKL breaks down myelin sheath in non-injured nerves d MLKL-mediated myelin breakdown promotes functional axon regeneration

In response to apoptotic stimuli, mitochondria in mammalian cells release cytochrome c and other apoptogenic proteins, leading to the subsequent activation of caspases and apoptotic cell death. This process is promoted by the pro-apoptotic members of the Bcl-2 family of proteins, such as Bim and Bax, which, respectively, initiate and execute cytochrome c release from the mitochondria. Here we report the discovery of a small molecule that efficiently blocks Bim-induced apoptosis after Bax is activated on the mitochondria. The cellular target of this small molecule was identified to be the succinate dehydrogenase subunit B (SDHB) protein of complex II of the mitochondrial electron transfer chain (ETC). The molecule protects the integrity of the ETC and allows treated cells to continue to proliferate after apoptosis induction. Moreover, this molecule blocked dopaminergic neuron death and reversed Parkinson-like behavior in a rat model of Parkinson's disease.

Significance Peripheral nerve injury often occurs at axonal sites remote from the neuronal cell bodies. Information about these remote injuries must be accurately relayed back to the cell body as part of the reprogramming that occurs to transition the neuron to a regenerating state. We provide new insights into how the axonal endoplasmic reticulum (ER) proximal to the injury site generates a critical regeneration signal in sensory neurons. This involves injury-triggered activation and synthesis of an intraaxonal, ER transmembrane transcription factor, Luman/cAMP response element binding protein 3, that is retrogradely transported back into the nucleus. Manipulation of axon-derived Luman expression in injured sensory neurons reveals an important role for Luman in regulation of the intrinsic elongating form of axonal growth associated with the regeneration state.

We recently revealed that the axon endoplasmic reticulum resident transcription factor Luman/CREB3 (herein called Luman) serves as a unique retrograde injury signal in regulation of the intrinsic elongating form of sensory axon regeneration. Here, evidence supports that Luman contributes to axonal regeneration through regulation of the unfolded protein response (UPR) and cholesterol biosynthesis in adult rat sensory neurons. One day sciatic nerve crush injury triggered a robust increase in UPR-associated mRNA and protein expression in both neuronal cell bodies and the injured axons. Knockdown of Luman expression in 1 d injury-conditioned neurons by siRNA attenuated axonal outgrowth to 48% of control injured neurons and was concomitant with reduced UPR-and cholesterol biosynthesisassociated gene expression. UPR PCR-array analysis coupled with qRT-PCR identified and confirmed that four transcripts involved in cholesterol regulation were downregulated Ͼ2-fold by the Luman siRNA treatment of the injury-conditioned neurons. Further, the Luman siRNA-attenuated outgrowth could be significantly rescued by either cholesterol supplementation or 2 ng/ml of the UPR inducer tunicamycin, an amount determined to elevate the depressed UPR gene expression to a level equivalent of that observed with crush injury. Using these approaches, outgrowth increased significantly to 74% or 69% that of injury-conditioned controls, respectively. The identification of Luman as a regulator of the injury-induced UPR and cholesterol at levels that benefit the intrinsic ability of axotomized adult rat sensory neurons to undergo axonal regeneration reveals new therapeutic targets to bolster nerve repair.

Demyelination in the central nervous system (CNS) underlies many human diseases, including multiple sclerosis (MS). We report here the findings of our study of the CNS demyelination process using immune-induced [experimental autoimmune encephalomyelitis (EAE)] and chemical-induced [cuprizone (CPZ)] mouse models of demyelination. We found that necroptosis, a receptor-interacting protein 3 (RIP3) kinase and its substrate mixed lineage kinase domainlike protein (MLKL)-dependent cell death program, played no role in the demyelination process, whereas the MLKL-dependent, RIP3independent function of MLKL in the demyelination process initially discovered in the peripheral nervous system in response to nerve injury, also functions in demyelination in the CNS in these models. Moreover, a receptor-interacting protein 1 (RIP1) kinase inhibitor, RIPA-56, blocked disease progression in the EAE-induced model but showed no effect in the CPZ-induced model. It does so most likely at a step of monocyte elevation downstream of T cell activation and myelin-specific antibody generation, although upstream of breakdown of the blood-brain barrier. RIP1-kinase dead knock-in mice shared a similar result as mice treated with the RIP1 inhibitor. These results indicate that RIP1 kinase inhibitor is a potential therapeutic agent for immune-mediated demyelination diseases that works by prevention of monocyte elevation, a function previously unknown for RIP1 kinase.RIP1 kinase | myelin | demyelination | multiple sclerosis | MLKL M yelin is a lipid-rich (fatty) substance formed by glial cells called oligodendrocytes in the central nervous system (CNS) and by Schwann cells in the peripheral nervous system (PNS) that protects and nourishes neuron axons by wrapping the axons (1). Physiologically, the myelin structure speeds the transmission of electrical impulses called action potentials along myelinated axons by insulating the axon and reducing axonal membrane capacitance (2). The demyelination process occurs during nerve injury and happens in many human diseases, including, for example, in the PNS, Charcot-Marie-Tooth disease (3) and abdominal numbness brought about by diabetes (4), and in the CNS, multiple sclerosis (MS), during which the insulating covers of nerve cells in the brain and spinal cord are damaged by infiltrated immune cells, such as T cells and monocytes (5,6). Patients with MS are paralyzed gradually from the lower body to the upper body and eventually die; most of these patients are young adults ranging in age from their 20s-40s (7).The molecular mechanisms that underlie these demyelination diseases are largely unknown. A recent study indicated that preventing oligodendrocytes from necroptosis, a regulated form of necrotic cell death induced by ligands of the tumor necrosis factor (TNF) receptor family or Toll-like receptors, as well as certain viral infections, could decrease disease symptoms in MS disease models of experimental autoimmune encephalomyelitis (EAE) and a cuprizone (CPZ)-induced demyelination model (8). The TNF re...

Cells respond to perturbations in the microenvironment of the endoplasmic reticulum (ER), and to the overloading of its capacity to process secretory and membrane-associate proteins, by activating the Unfolded Protein Response (UPR). Genes that mediate the UPR are regulated by three basic leucine-zipper (bLZip) motif-containing transcription factors – Xbp1s, ATF4 and ATF6. A failure of the UPR to achieve homeostasis and its continued stimulation leads to apoptosis. Mechanisms must therefore exist to turn off the UPR if it successfully restores normalcy. The bLZip protein Zhangfei/CREBZF/SMILE is known to suppress the ability of several, seemingly structurally unrelated, transcription factors. These targets include Luman/CREB3 and CREBH, ER-resident bLZip proteins known to activate the UPR in some cell types. Here we show that Zhangfei had a suppressive effect on most UPR genes activated by the calcium ionophore thapsigargin. This effect was at least partially due to the interaction of Zhangfei with Xbp1s. The leucine zipper of Zhangfei was required for this interaction, which led to the subsequent proteasomal degradation of Xbp1s. Zhangfei suppressed the ability of Xbp1s to activate transcription from a promoter containing unfolded protein response elements and significantly reduced the ability to Xbp1s to activate the UPR as measured by RNA and protein levels of UPR-related genes. Finally, specific suppression of endogenous Zhangfei in thapsigargin-treated primary rat sensory neurons with siRNA directed to Zhangfei transcripts, led to a significant increase in transcripts and proteins of UPR genes, suggesting a potential role for Zhangfei in modulating the UPR.

No abstract

A series of 2-sulfonyl-pyrimidinyl derivatives was developed as apoptosis inhibitors. These represent the first class of apoptosis inhibitors that function through stabilizing mitochondrial respiratory complex II. Starting from a phenotypic screen hit with micromolar activity, we optimized the cellular apoptosis inhibition activity of 2-sulfonyl-pyrimidinyl derivatives to picomolar level (compound , also named as TC9-305). The therapeutic potential of these new apoptosis inhibitors was further demonstrated by their neuroprotective effect on an ischemic animal model.