Mixed Lineage Kinase Domain-like Protein MLKL Breaks Down Myelin following Nerve Injury.

Successful regeneration of severed peripheral nerves requires the breakdown and subsequent clearance of myelin, tightly packed membrane sheaths of Schwann cells that protect nerve fibers and harbor nerve growth-inhibitory proteins. How Schwann cells initiate myelin breakdown in response to injury is still largely unknown. Here we report that, following sciatic nerve injury, MLKL, a pseudokinase known to rupture cell membranes during necroptotic cell death, is induced and targets the myelin sheath membrane of Schwann cells to promote myelin breakdown. The function of MLKL in disrupting myelin sheaths requires injury-induced phosphorylation of serine 441, an activation signal distinct from the necroptosis-inducing phosphorylation by RIP3 kinase. Mice with Mlkl specifically knocked out in Schwann cells showed delayed myelin sheath breakdown. Lack of MLKL reduced nerve regeneration following injury, whereas overexpression of MLKL accelerated myelin breakdown and promoted the regeneration of axons.

Blockage of MLKL prevents myelin damage in experimental diabetic neuropathy.

SignificanceDiabetic neuropathy is a commonly occurring complication of diabetes that affects hundreds of millions of patients worldwide. Patients suffering from diabetic neuropathy experience abnormal sensations and have damage in their peripheral nerve axons as well as myelin, a tightly packed Schwann cell sheath that wraps around axons to provide insulation and increases electrical conductivity along the nerve fibers. The molecular events underlying myelin damage in diabetic neuropathy are largely unknown, and there is no efficacious treatment for the disease. The current study, using a diabetic mouse model and human patient nerve samples, uncovered a molecular mechanism underlying myelin sheath damage in diabetic neuropathy and provides a potential treatment strategy for the disease.

A Small Molecule That Protects the Integrity of the Electron Transfer Chain Blocks the Mitochondrial Apoptotic Pathway.

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.

A small molecule protects mitochondrial integrity by inhibiting mTOR activity.

Apoptosis activation by cytochrome c release from mitochondria to cytosol is a normal cellular response to mitochondrial damage. Using cellular apoptosis assay, we have found small-molecule apoptosis inhibitors that protect cells from mitochondrial damage. Previously, we reported the discovery of a small molecule, Compound A, which blocks dopaminergic neuron death in a rat model of Parkinson's disease through targeting succinate dehydrogenase subunit B (SDHB) of complex II to protect the integrity of the mitochondrial respiratory chain. Here, we report a small molecule, Compound R6, which saves cells from apoptosis via mammalian target of rapamycin (mTOR)-mediated induction of autophagy. Additionally, we show that Compound R6 protects mitochondrial integrity and respiration after induction of the intrinsic apoptosis pathway. Encouragingly, and supporting the potential further application of Compound R6 as a tool for basic and medicinal research, a pharmacokinetics (PK) profiling study showed that Compound R6 is metabolically stable and can pass the blood-brain barrier. Moreover, Compound R6 accumulates in the brain of test animals via intravenous and intraperitoneal administration. Finally, we found that Compound R6 confers significant neuroprotective effects on a rat cerebral ischemia/reperfusion model, demonstrating its potential as a promising drug candidate for neurodegenerative diseases.

RIP1 kinase inhibitor halts the progression of an immune-induced demyelination disease at the stage of monocyte elevation.

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 domain-like protein (MLKL)-dependent cell death program, played no role in the demyelination process, whereas the MLKL-dependent, RIP3-independent 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.

Sensing nerve injury at the axonal ER: activated Luman/CREB3 serves as a novel axonally synthesized retrograde regeneration signal.

Luman/cAMP response element binding protein 3 is an endoplasmic reticulum (ER) transmembrane basic leucine zipper transcription factor whose mRNA and protein localize to adult sensory axons, the latter with axonal ER components along the axon length. Here we show that axon-derived Luman plays an important role in relaying information about axonal injury to the neuronal cell body. Axotomy induces axonal Luman synthesis and also release from the axonal ER of Luman's transcriptionally active amino terminus, which is transported to the cell body in an importin-mediated manner. Visualization of the activation and retrograde translocation of Luman into the nucleus in real time both in vivo and in vitro was accomplished using a specially created N- and C-terminal-tagged Luman adenoviral vector. Small interfering RNA used to reduce Luman expression either neuronally or just axonally significantly impaired the ability of 24-h injury-conditioned sensory neurons to extend the regeneration-associated elongating form of axon growth but had no impact on axon outgrowth in naïve neurons. Collectively, these findings link injury-associated axonal ER responses proximal to the site of injury to the intrinsic regenerative growth capacity of adult sensory neurons.

The Unfolded Protein Response and Cholesterol Biosynthesis Link Luman/CREB3 to Regenerative Axon Growth in Sensory Neurons.

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 biosynthesis-associated gene expression. UPR PCR-array analysis coupled with qRT-PCR identified and confirmed that four transcripts involved in cholesterol regulation were downregulated >2-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.

Extracellular pH and neuronal depolarization serve as dynamic switches to rapidly mobilize trkA to the membrane of adult sensory neurons.

Activation of the nerve growth factor (NGF) receptor trkA and tissue acidosis are critically linked to inflammation-associated nociceptor sensitization. This study explored how increased acidity is linked to sensory neuron sensitization to NGF. Adult Wistar rat primary sensory neurons grown at physiological pH 7.4, then either kept at pH 7.4 or challenged for 30 min in pH 6.5 medium, provided a model of acidosis. Nonpermeabilizing trkA immunofluorescence revealed a significant increase in trkA mobilization to the plasma membrane from intracellular stores in response to proton challenge. This was confirmed using a surface protein biotinylation assay and Brefeldin A disruption of the rough endoplasmic reticulum-Golgi-trans-Golgi network. Mobilization of trkA to the membrane at pH 6.5 was abolished in neurons treated with the acid-sensitive ion channel blocker, amiloride. While elevated levels of NGF-independent trkA phosphorylation occurred at pH 6.5 alone, the level of activation was significantly increased in response to NGF challenge. Exposure of sensory neurons to pH 6.5 medium also resulted in strong calcium (Ca(2+)) transients that were reversible upon reintroduction to physiological pH. The pH 6.5-induced mobilization of trkA to the membrane was Ca(2+) dependent, as BAPTA-AM Ca(2+) chelation abrogated the response. Interestingly, KCl-induced depolarization was sufficient to induce mobilization of trkA to the cell surface at pH 7.4, but did not augment the response to pH 6.5. In conclusion, increased mobilization of trkA to neuronal membranes in response to either acidosis or neuronal depolarization provides two novel mechanisms by which sensory neurons can rapidly sensitize to NGF and has important implications for inflammatory pain states.

Schwann cell promotes macrophage recruitment through IL-17B/IL-17RB pathway in injured peripheral nerves.

Macrophage recruitment to the injured nerve initiates a cascade of events, including myelin debris clearance and nerve trophic factor secretion, which contribute to proper nerve tissue repair. However, the mechanism of macrophage recruitment is still unclear. Here, by comparing wild-type with Mlkl-/- and Sarm1-/- mice, two mouse strains with impaired myelin debris clearance after peripheral nerve injury, we identify interleukin-17B (IL-17B) as a key regulator of macrophage recruitment. Schwann-cell-secreted IL-17B acts in an autocrine manner and binds to IL-17 receptor B to promote macrophage recruitment, and global or Schwann-cell-specific IL-17B deletion reduces macrophage infiltration, myelin clearance, and axon regeneration. We also show that the IL-17B signaling pathway is defective in the injured central nerves. These results reveal an important role for Schwann cell autocrine signaling during Wallerian degeneration and point to potential mechanistic targets for accelerating myelin clearance and improving demyelinating disease.

Spatial localization ability of planarians identified through a light maze paradigm.

Spatial localization ability is crucial for free-living animals to fit the environment. As shown by previous studies, planarians can be conditioned to discriminate directions. However, due to their simplicity and primitiveness, they had never been considered to have true spatial localization ability to retrieve locations of objects and places in the environment. Here, we introduce a light maze training paradigm to demonstrate that a planarian worm can navigate to a former recognized place from the start point, even if the worm is transferred into a newly produced maze. This finding identifies the spatial localization ability of planarians for the first time, which provides clues for the evolution of spatial learning. Since the planarians have a primitive brain with simple structures, this paradigm can also provide a simplified model for a detailed investigation of spatial learning.

Structural and functional insights of the human peroxisomal ABC transporter ALDP.

Adrenoleukodystrophy protein (ALDP) is responsible for the transport of very-long-chain fatty acids (VLCFAs) and corresponding CoA-esters across the peroxisomal membrane. Dysfunction of ALDP leads to peroxisomal metabolic disorder exemplified by X-linked adrenoleukodystrophy (ALD). Hundreds of ALD-causing mutations have been identified on ALDP. However, the pathogenic mechanisms of these mutations are restricted to clinical description due to limited structural and biochemical characterization. Here we report the cryo-electron microscopy structure of human ALDP with nominal resolution at 3.4 Å. ALDP exhibits a cytosolic-facing conformation. Compared to other lipid ATP-binding cassette transporters, ALDP has two substrate binding cavities formed by the transmembrane domains. Such structural organization may be suitable for the coordination of VLCFAs. Based on the structure, we performed integrative analysis of the cellular trafficking, protein thermostability, ATP hydrolysis, and the transport activity of representative mutations. These results provide a framework for understanding the working mechanism of ALDP and pathogenic roles of disease-associated mutations.

α-Synuclein increases U251 cells vulnerability to hydrogen peroxide by disrupting calcium homeostasis.

Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by glial cytoplasmic inclusions containing insoluble α-synuclein. Since Ca(2+) plays an important role in cell degeneration, [Ca(2+)]( i ) in α-synuclein-overexpressed human glioma cells was analyzed by Fura-2 fluorometry. Overexpression of α-synuclein increased the basal level of [Ca(2+)]( i ), and a higher Ca(2+) response to hydrogen peroxide was further observed. The effect that α-synuclein overexpression caused U251 cells to be more vulnerable to hydrogen peroxide was eliminated by Ca(2+) chelator BAPTA-AM or transient receptor potential channels blocker SKF 96365 but not by L-type Ca(2+) channel blocker nimodipine. These findings suggest that the dysregulation of cellular Ca(2+) homeostasis caused by α-synuclein under oxidative stress may contribute to the glial cell death in MSA.

α-Synuclein binds to cytoplasmic vesicles in U251 glioblastoma cells.

α-Synuclein is the major component of Lewy bodies, Lewy neurites, and glial cytoplasmic inclusions. It plays an important role in neurodegenerative diseases such as Parkinson's disease, multiple system atrophy, and other synucleinopathies. However, the pathogenesis and neurodegenerative effects of α-synuclein remain unknown. In this study, we established an α-synuclein and an α-synuclein-EGFP overexpressing U251 cell line. α-Synuclein overexpression increases oxidative stress and alters the cell surface and mitochondrial morphologies. We provide fluorescent-protein tagging, immunofluorescence and ultrastructural evidence showing that α-synuclein accumulations are associated with clusters of cytoplasmic vesicles and the diameter of these vesicles increases by H2O2 in a time- and dose-dependent manner.

Discovery of Highly Potent 2-Sulfonyl-Pyrimidinyl Derivatives for Apoptosis Inhibition and Ischemia Treatment.

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 42, 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.

Cloning and characterization of rat Luman/CREB3, a transcription factor highly expressed in nervous system tissue.

Human Luman/CREB3 is a basic leucine zipper transcription factor involved in regulation of the unfolded protein response, dendritic cell maturation, and cell migration. But despite reported expression in primary sensory neurons, little is known about its role in the nervous system. To begin investigations into its role in the adult rat nervous system, the rat Luman/CREB3 coding sequence was isolated so its expression within the nervous system could be determined. The rat Luman/CREB3 clone contains a full-length open reading frame encoding 387 amino acids. The recombinant protein generated from this clone activated transcription in a manner equivalent to human Luman/CREB3 from a CAT reporter plasmid construct containing the unfolded protein response element. Quantitative RT-PCR revealed that rat Luman/CREB3 transcripts in a variety of rat tissues with the highest levels in nervous system tissue. In situ hybridization performed on tissue sections confirmed the findings and demonstrated that the Luman/CREB3 mRNA hybridization signal localizes to neurons and satellite glial cells in dorsal root ganglia, the cytoplasm of hepatocytes in liver, and the hippocampal pyramidal cell layers of CA1 and CA3 and the granular cell layer of the dentate gyrus. Collectively, these findings support a role for Luman/CREB3 in the regulation of nervous system function.

Injury-associated PACAP expression in rat sensory and motor neurons is induced by endogenous BDNF.

Peripheral nerve injury results in dramatic upregulation in pituitary adenylate cyclase activating polypeptide (PACAP) expression in adult rat dorsal root ganglia and spinal motor neurons mirroring that described for the neurotrophin brain derived neurotrophic factor (BDNF). Thus, we posited that injury-associated alterations in BDNF expression regulate the changes in PACAP expression observed in the injured neurons. The role of endogenous BDNF in induction and/or maintenance of PACAP mRNA expression in injured adult rat motor and sensory neurons was examined by intrathecally infusing or intraperitoneally injecting BDNF-specific antibodies or control IgGs immediately at the time of L4-L6 spinal nerve injury, or in a delayed fashion one week later for 3 days followed by analysis of impact on PACAP expression. PACAP mRNA in injured lumbar sensory and motor neurons was detected using in situ hybridization, allowing quantification of relative changes between experimental groups, with ATF-3 immunofluorescence serving to identify the injured subpopulation of motor neurons. Both the incidence and level of PACAP mRNA expression were dramatically reduced in injured sensory and motor neurons in response to immediate intrathecal anti-BDNF treatment. In contrast, neither intraperitoneal injections nor delayed intrathecal infusions of anti-BDNF had any discernible impact on PACAP expression. This impact on PACAP expression in response to BDNF immunoneutralization in DRG was confirmed using qRT-PCR or by using BDNF selective siRNAs to reduce neuronal BDNF expression. Collectively, our findings support that endogenous injury-associated BDNF expression is critically involved in induction, but not maintenance, of injury-associated PACAP expression in sensory and motor neurons.

Increased vulnerability of parkin knock down PC12 cells to hydrogen peroxide toxicity: the role of salsolinol and NM-salsolinol.

Dopamine-derived neurotoxins, 1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) and 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (NM-salsolinol) are the two most possible 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-like endogenous neurotoxin candidates that involved in the pathogenesis of Parkinson's disease (PD). The levels of endogenously synthesized salsolinol and NM-salsolinol are increased in the cerebrospinal fluid (CSF) of PD patients. Both of them lead to neurotoxicity in dopaminergic cells by inhibiting mitochondrial electron transport chain. To study the role of salsolinol and NM-salsolinol in Parkin deficiency-induced dopaminergic cell damage, we determined the cellular level of oxidative stress, the formation of salsolinol and NM-salsolinol, the level of mitochondrial damage and cell viability with/without the presence of exogenous H2O2 using differentiated dopaminergic PC12 cells. Our data show that parkin knock down elevates cellular oxidative stress, salsolinol and NM-salsolinol levels, which are responsible for the higher cell mortality in Parkin-deficient cells upon exposure to exogenous H2O2. The level of mitochondrial membrane potential loss, cristae disruption and the release of cytochrome c increased significantly along with the increased level of salsolinol and NM-salsolinol, whereas compared to parkin knock down cells in the presence of H2O2, the mitochondrial damage and higher cell mortality were both diminished when the levels of salsolinol and NM-salsolinol was reduced. The results not only indicate the elevated level of salsolinol and NM-salsolinol, but also reveal the potential role of salsolinol and NM-salsolinol in parkin knock down-induced cell vulnerability. We assume that parkin deficiency is the trigger of excessive oxidative stress, elevated endogenous neurotoxin levels and mitochondrial damage, which eventually results in cell death of dopaminergic cells.

Zhangfei/CREB-ZF - a potential regulator of the unfolded protein response.

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.

Lys(203) and Lys(382) are essential for the proteasomal degradation of BACE1.

Amyloid ß protein (Aß) is the primary component of senile plaques in Alzheimer's disease brains and its aggregate form is neurotoxic. Aß is generated through proteolysis of ß-amyloid precursor protein (APP) by two proteases: ß-secretase and γ-secretase. BACE1, the ß-secretase in vivo and the key rate-limiting enzyme that initiates the formation of Aß, is an attractive drug target for AD therapy. Our previous study demonstrated that BACE1 is ubiquitinated and its degradation and effect on APP cleaving process are mediated by the ubiquitin-proteasome pathway. However, the specific underlying mechanism is still not well defined. In present study, we determined the specific binding sites responsible for the proteasomal degradation of BACE1. Ten fragments of human BACE1 cDNA with each of them containing 1 to 3 Lys codons were cloned, and HEK293 cells transfected with these recombinant plasmids were treated with specific proteasome inhibitor lactacystin. The protein levels of fragment-3 (Pro(149)-Leu(180)), -4 (IIe(179)-Ser(230)) and -8 (Met(349)-Arg(400)) were significantly increased by lactacystin treatment, and immunocytochemical staining results showed that fragment-3, -4 and -8 proteins were colocalized with ubiquitin. Site-directed mutagenesis at Lys(203) and Lys(382) of BACE1 abolished the proteasomal degradation of BACE1 and affected APP processing at ß site and Aß production. Taken together, our study demonstrated that BACE1 Lys(203) and Lys(382) are essential for its proteasomal degradation, and the results may advance our understanding of regulation of BACE1 and APP processing by the ubiquitin proteasome system in AD pathogenesis and shed new insights on its pharmaceutical potential.