Rapamycin improves peripheral nerve myelination while it fails to benefit neuromuscular performance in neuropathic mice.

Charcot--Marie-Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy characterized by progressive demyelination and distal muscle weakness. Abnormal expression of peripheral myelin protein 22 (PMP22) has been linked to CMT1A and is modeled by Trembler J (TrJ) mice, which carry the same leucine to proline substitution in PMP22 as affected pedigrees. Pharmacologic modulation of autophagy by rapamycin in neuron-Schwann cell explant cultures from neuropathic mice reduced PMP22 aggregate formation and improved myelination. Here we asked whether rapamycin administration by food supplementation, or intraperitoneal injection, could alleviate the neuropathic phenotype of affected mice and improve neuromuscular performance. Cohorts of male and female wild type (Wt) and TrJ mice were assigned to placebo or rapamycin treatment starting at 2 or 4months of age and tested monthly on the rotarod. While neither long-term feeding (8 or 10months) on rapamycin-enriched diet, or short-term injection (2months) of rapamycin improved locomotor performance of the neuropathic mice, both regimen benefited peripheral nerve myelination. Together, these results indicate that while treatment with rapamycin benefits the myelination capacity of neuropathic Schwann cells, this intervention does not improve neuromuscular function. The observed outcome might be the result of the differential response of nerve and skeletal muscle tissue to rapamycin.

Surgical procedure of intratympanic injection and inner ear pharmacokinetics simulation in domestic pigs.

Introduction: One major obstacle in validating drugs for the treatment or prevention of hearing loss is the limited data available on the distribution and concentration of drugs in the human inner ear. Although small animal models offer some insights into inner ear pharmacokinetics, their smaller organ size and different barrier (round window membrane) permeabilities compared to humans can complicate study interpretation. Therefore, developing a reliable large animal model for inner ear drug delivery is crucial. The inner and middle ear anatomy of domestic pigs closely resembles that of humans, making them promising candidates for studying inner ear pharmacokinetics. However, unlike humans, the anatomical orientation and tortuosity of the porcine external ear canal frustrates local drug delivery to the inner ear. Methods: In this study, we developed a surgical technique to access the tympanic membrane of pigs. To assess hearing pre- and post-surgery, auditory brainstem responses to click and pure tones were measured. Additionally, we performed 3D segmentation of the porcine inner ear images and used this data to simulate the diffusion of dexamethasone within the inner ear through fluid simulation software (FluidSim). Results: We have successfully delivered dexamethasone and dexamethasone sodium phosphate to the porcine inner ear via the intratympanic injection. The recorded auditory brainstem measurements revealed no adverse effects on hearing thresholds attributable to the surgery. We have also simulated the diffusion rates for dexamethasone and dexamethasone sodium phosphate into the porcine inner ear and confirmed the accuracy of the simulations using in-vivo data. Discussion: We have developed and characterized a method for conducting pharmacokinetic studies of the inner ear using pigs. This animal model closely mirrors the size of the human cochlea and the thickness of its barriers. The diffusion time and drug concentrations we reported align closely with the limited data available from human studies. Therefore, we have demonstrated the potential of using pigs as a large animal model for studying inner ear pharmacokinetics.

Amino acid homeostasis and chronological longevity in Saccharomyces cerevisiae.

Understanding how non-dividing cells remain viable over long periods of time, which may be decades in humans, is of central importance in understanding mechanisms of aging and longevity. The long-term viability of non-dividing cells, known as chronological longevity, relies on cellular processes that degrade old components and replace them with new ones. Key among these processes is amino acid homeostasis. Amino acid homeostasis requires three principal functions: amino acid uptake, de novo synthesis, and recycling. Autophagy plays a key role in recycling amino acids and other metabolic building blocks, while at the same time removing damaged cellular components such as mitochondria and other organelles. Regulation of amino acid homeostasis and autophagy is accomplished by a complex web of pathways that interact because of the functional overlap at the level of recycling. It is becoming increasingly clear that amino acid homeostasis and autophagy play important roles in chronological longevity in yeast and higher organisms. Our goal in this chapter is to focus on mechanisms and pathways that link amino acid homeostasis, autophagy, and chronological longevity in yeast, and explore their relevance to aging and longevity in higher eukaryotes.

Cerebrospinal Fluid Flow Extends to Peripheral Nerves.

Cerebrospinal fluid (CSF) is an aqueous solution responsible for nutrient delivery and waste removal for the central nervous system (CNS). The three-layer meningeal coverings of the CNS support CSF flow. Peripheral nerves have an analogous three-layer covering consisting of the epineurium, perineurium, and endoneurium. Peripheral axons, located in the inner endoneurium, are bathed in "endoneurial fluid" similar to CSF but of undefined origin. CSF flow in the peripheral nervous system has not been demonstrated. Here we show CSF flow extends beyond the CNS to peripheral nerves in a contiguous flowing system. Utilizing gold nanoparticles, we identified that CSF is continuous with the endoneurial fluid and reveal the endoneurial space as the likely site of CSF flow in the periphery. Nanogold distribution along entire peripheral nerves and within their axoplasm suggests CSF plays a role in nutrient delivery and waste clearance, fundamental aspects of peripheral nerve health and disease. One Sentence Summary: Cerebrospinal fluid unites the nervous system by extending beyond the central nervous system into peripheral nerves.

Autophagy suppresses age-dependent ischemia and reperfusion injury in livers of mice.

BACKGROUND & AIMS: As life expectancy increases, there are greater numbers of patients with liver diseases who require surgery or transplantation. Livers of older patients have significantly less reparative capacity following ischemia and reperfusion (I/R) injury, which occurs during these operations. There are no strategies to reduce the age-dependent I/R injury. We investigated the role of autophagy in the age dependence of sensitivity to I/R injury. METHODS: Hepatocytes and livers from 3- and 26-month-old mice were subjected to in vitro and in vivo I/R, respectively. We analyzed changes in autophagy-related proteins (Atg). Mitochondrial dysfunction was visualized using confocal and intravital multi-photon microscopy of isolated hepatocytes and livers from anesthetized mice, respectively. RESULTS: Immunoblot, autophagic flux, genetic, and imaging analyses all associated the increase in sensitivity to I/R injury with age with decreased autophagy and subsequent mitochondrial dysfunction due to calpain-mediated loss of Atg4B. Overexpression of either Atg4B or Beclin-1 recovered Atg4B, increased autophagy, blocked the onset of the mitochondrial permeability transition, and suppressed cell death after I/R in old hepatocytes. Coimmunoprecipitation analysis of hepatocytes and Atg3-knockout cells showed an interaction between Beclin-1 and Atg3, a protein required for autophagosome formation. Intravital multi-photon imaging revealed that overexpression of Beclin-1 or Atg4B attenuated autophagic defects and mitochondrial dysfunction in livers of older mice after I/R. CONCLUSIONS: Loss of Atg4B in livers of old mice increases their sensitivity to I/R injury. Increasing autophagy might ameliorate liver damage and restore mitochondrial function after I/R.

Rapamycin activates autophagy and improves myelination in explant cultures from neuropathic mice.

Misexpression and cytosolic retention of peripheral myelin protein 22 (PMP22) within Schwann cells (SCs) is associated with a genetically heterogeneous group of demyelinating peripheral neuropathies. PMP22 overproducer C22 and spontaneous mutant Trembler J (TrJ) mice display neuropathic phenotypes and affected nerves contain abnormally localized PMP22. Nutrient deprivation-induced autophagy is able to suppress the formation of PMP22 aggregates in a toxin-induced cellular model, and improve locomotor performance and myelination in TrJ mice. As a step toward therapies, we assessed whether pharmacological activation of autophagy by rapamycin (RM) could facilitate the processing of PMP22 within neuropathic SCs and enhance their capacity to myelinate peripheral axons. Exposure of mouse SCs to RM induced autophagy in a dose- and time-dependent manner and decreased the accumulation of poly-ubiquitinated substrates. The treatment of myelinating dorsal root ganglion (DRG) explant cultures from neuropathic mice with RM (25 nm) improved the processing of PMP22 and increased the abundance and length of myelin internodes, as well as the expression of myelin proteins. Notably, RM is similarly effective in both the C22 and TrJ model, signifying that the benefit overlaps among distinct genetic models of PMP22 neuropathies. Furthermore, lentivirus-mediated shRNA knockdown of the autophagy-related gene 12 (Atg12) abolished the activation of autophagy and the increase in myelin proteins, demonstrating that autophagy is critical for the observed improvement. Together, these results support the potential use of RM and other autophagy-enhancing compounds as therapeutic agents for PMP22-associated demyelinating neuropathies.

Intermittent fasting alleviates the neuropathic phenotype in a mouse model of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth type 1A (CMT1A) neuropathies linked to the misexpression of peripheral myelin protein 22 (PMP22) are progressive demyelinating disorders of the peripheral nervous system. In this study we asked whether dietary restriction by intermittent fasting (IF) could alleviate the neuropathic phenotype in the Trembler J (TrJ) mouse model of CMT1A. Our results show that neuropathic mice kept on a five month long IF regimen had improved locomotor performance compared to ad libitum (AL) fed littermates. The functional benefits of this dietary intervention are associated with an increased expression of myelin proteins combined with a thicker myelin sheath, less redundant basal lamina, and a reduction in aberrant Schwann cell proliferation. These morphological improvements are accompanied by a decrease in PMP22 protein aggregates, and enhanced expression of cytosolic chaperones and constituents of the autophagy-lysosomal pathway. These results indicate that dietary restriction is beneficial for peripheral nerve function in TrJ neuropathic mice, as it promotes the maintenance of locomotor performance.

Impaired autophagy: A mechanism of mitochondrial dysfunction in anoxic rat hepatocytes.

UNLABELLED: Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of autophagy-related protein 7 (Atg7), Beclin-1, and Atg12, autophagy regulatory proteins, was analyzed by western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding green fluorescent protein (control), Atg7, and Beclin-1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in an amino acid-free and serum-free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were coloaded with calcein and tetramethylrhodamine methyl ester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with an adenovirus expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) and subjected to A/R. Calpain activity was fluorometrically determined with succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. A/R markedly decreased Atg7 and Beclin-1 concomitantly with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression, and nutrient depletion all substantially suppressed A/R-induced loss of autophagy proteins, prevented onset of the MPT, and decreased cell death after reoxygenation. Confocal imaging of GFP-LC3 confirmed A/R-induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. CONCLUSION: Calpain 2-mediated degradation of Atg7 and Beclin-1 impairs mitochondrial autophagy, and this subsequently leads to MPT-dependent hepatocyte death after A/R.

Immunologic Alterations Associated With Oral Delivery of Anti-CD3 (OKT3) Monoclonal Antibodies in Patients With Moderate-to-Severe Ulcerative Colitis.

AIM: The aim of this study was to determine the immunologic effects and safety of oral anti-CD3 in patients with ulcerative colitis (UC). METHODS: An open-label pilot study of orally delivered anti-CD3 was performed in patients with moderate-to-severe UC. The primary end points were changes in immunologic parameters and evaluation for safety. RESULTS: Six subjects received oral OKT3. Biologic effects of oral anti-CD3 included significantly increased proliferation in response to anti-CD3 and anti-inflammatory gene expression profile in peripheral blood mononuclear cells. No serious treatment-related adverse events occurred. CONCLUSION: Orally delivered anti-CD3 resulted in immunologic changes in patients with UC.

Changes in autophagy proteins in a rat model of controlled cortical impact induced brain injury.

Autophagy has been implicated in several neurodegenerative diseases and recently its role in acute brain injury has received increased interest. In our study, we investigated the profiles of autophagy-linked proteins (MAP-LC3 (Atg8), beclin-1 (Atg6) and the beclin-1-binding protein, bcl-2, following controlled cortical impact injury in rats--a model for moderate-to-severe traumatic brain injury. We observed significant increases in the levels of the processed form of LC3 (LC3-II) in the ipsilateral cortex 2h to 2 days after injury when compared to sham. Furthermore, the beclin-1/bcl-2 ratio in the ipsilateral cortex was found to have increased from 1 and 2 days after injury. Since both of these changes are established autophagy-enabling events, and, based on these data, we propose that autophagy, plays a role in the manifestation of cell injury following brain trauma.

PpATG9 encodes a novel membrane protein that traffics to vacuolar membranes, which sequester peroxisomes during pexophagy in Pichia pastoris.

When Pichia pastoris adapts from methanol to glucose growth, peroxisomes are rapidly sequestered and degraded within the vacuole by micropexophagy. During micropexophagy, sequestering membranes arise from the vacuole to engulf the peroxisomes. Fusion of the sequestering membranes and incorporation of the peroxisomes into the vacuole is mediated by the micropexophagy-specific membrane apparatus (MIPA). In this study, we show the P. pastoris ortholog of Atg9, a novel membrane protein is essential for the formation of the sequestering membranes and assembly of MIPA. During methanol growth, GFP-PpAtg9 localizes to multiple structures situated near the plasma membrane referred as the peripheral compartment (Atg9-PC). On glucose-induced micropexophagy, PpAtg9 traffics from the Atg9-PC to unique perivacuolar structures (PVS) that contain PpAtg11, but lack PpAtg2 and PpAtg8. Afterward, PpAtg9 distributes to the vacuole surface and sequestering membranes. Movement of the PpAtg9 from the Atg9-PC to the PVS requires PpAtg11 and PpVps15. PpAtg2 and PpAtg7 are essential for PpAtg9 trafficking from the PVS to the vacuole and sequestering membranes, whereas trafficking of PpAtg9 proceeds independent of PpAtg1, PpAtg18, and PpVac8. In summary, our data suggest that PpAtg9 transits from the Atg9-PC to the PVS and then to the sequestering membranes that engulf the peroxisomes for degradation.

Peripheral myelin protein 22 is in complex with alpha6beta4 integrin, and its absence alters the Schwann cell basal lamina.

Peripheral myelin protein 22 (PMP22) is a tetraspan membrane glycoprotein, the misexpression of which is associated with hereditary demyelinating neuropathies. Myelinating Schwann cells (SCs) produce the highest levels of PMP22, yet the function of the protein in peripheral nerve biology is unresolved. To investigate the potential roles of PMP22, we engineered a novel knock-out (-/-) mouse line by replacing the first two coding exons of pmp22 with the lacZ reporter. PMP22-deficient mice show strong beta-galactosidase reactivity in peripheral nerves, cartilage, intestines, and lungs, whereas phenotypically they display the characteristics of tomaculous neuropathy. In the absence of PMP22, myelination of peripheral nerves is delayed, and numerous axon-SC profiles show loose basal lamina, suggesting altered interactions of the glial cells with the extracellular matrix. The levels of beta4 integrin, a molecule involved in the linkage between SCs and the basal lamina, are severely reduced in nerves of PMP22-deficient mice. During early stages of myelination, PMP22 and beta4 integrin are coexpressed at the cell surface and can be coimmunoprecipitated together with laminin and alpha6 integrin. In agreement, in clone A colonic carcinoma cells, epitope-tagged PMP22 forms a complex with beta4 integrin. Together, these data indicate that PMP22 is a binding partner in the integrin/laminin complex and is involved in mediating the interaction of SCs with the extracellular environment.

Autophagy in the heart and liver during normal aging and calorie restriction.

Autophagy is a highly regulated intracellular process for the degradation of cellular constituents and essential for the maintenance of a healthy cell. We evaluated the effects of age and life-long calorie restriction on autophagy in heart and liver of young (6 months) and old (26 months) Fisher 344 rats. We observed that the occurrence of autophagic vacuoles was higher in heart than liver. The occurrence of autophagic vacuoles was not affected by age in either tissue, but was increased with calorie restriction in heart but not in liver. Next, we examined the expression of proteins involved in the formation and maturation of autophagosomes (beclin-1, LC3, Atg7, Atg9) or associated with autolysosomes and lysosomes (LAMP-1; cathepsin D). In hearts of both ad libitum-fed and calorie-restricted rats, we observed an increase in expression of beclin-1 and procathepsin D, but not mature cathepsin D, and a decrease in expression of LAMP-1 because of aging. In hearts, calorie restriction stimulated the expression of Atg7 and Atg9 and the lipidation of Atg8 (elevated LC3-II/I ratios) in aged rats. In hearts of ad libitum-fed rats, expression of Atg7 and lipidation of Atg8 were unaffected by age, while the cellular levels of Atg9 were lower in aged animals. Furthermore, we observed that the age- and diet-dependent expression levels of those proteins differed between heart and liver. In conclusion, autophagy in heart and liver did not decrease with age in ad libitum-fed rats, but was enhanced by calorie restriction in the heart. Thus, calorie restriction may mediate some of its beneficial effects by stimulating autophagy in the heart, indicating the potential for cardioprotective therapies.

Identification of pexophagy genes by restriction enzyme-mediated integration.

Pichia pastoris has proven to be a valuable model for examining the molecular events of the selective degradation of peroxisomes by a process called pexophagy. We have developed a protocol to rapidly identify genes essential for glucose-induced pexophagy. This method utilizes the random integration of a Zeocin resistance cassette vector into the genomic DNA thereby disrupting gene expression. Transformed yeast are selected by growth on Zeocin and pexophagy mutants identified by their inability to degrade the peroxisomal enzyme alcohol oxidase. The Zeocin vector along with flanking genomic DNA is then isolated from the mutants and the disrupted genes identified by sequencing. We have been able to isolate 59 mutants and identify 8 unique genes. The identification of 24 genes in P. pastoris and 7 genes in H. polymorpha have been reported using this approach which has been referred to as restriction-mediated integration.

Chemical genetic analysis of Apg1 reveals a non-kinase role in the induction of autophagy.

Macroautophagy is a catabolic membrane trafficking phenomenon that is observed in all eukaryotic cells in response to various stimuli, such as nitrogen starvation and challenge with specific hormones. In the yeast Saccharomyces cerevisiae, the induction of autophagy involves a direct signal transduction mechanism that affects membrane dynamics. In this system, the induction process modifies a constitutive trafficking pathway called the cytoplasm-to-vacuole targeting (Cvt) pathway, which transports the vacuolar hydrolase aminopeptidase I, from the formation of small Cvt vesicles to the formation of autophagosomes. Apg1 is one of the proteins required for the direct signal transduction cascade that modifies membrane dynamics. Although Apg1 is required for both the Cvt pathway and autophagy, we find that Apg1 kinase activity is required only for Cvt trafficking of aminopeptidase I but not for import via autophagy. In addition, the data support a novel role for Apg1 in nucleation of autophagosomes that is distinct from its catalytic kinase activity and imply a qualitative difference in the mechanism of autophagosome and Cvt vesicle formation.

"Bath Salts" the New York City Medical Examiner Experience: A 3-Year Retrospective Review.

"Bath salts" are synthetic derivatives of cathinones, compounds found in the leaves of Catha edulis, which possesses amphetamine-like properties. At the New York City Office of Chief Medical Examiner, we conducted a 3-year retrospective analysis of deaths in which cathinones were detected. Two categories emerged; those in which cathinones were a contributory cause of death (15 cases) and those in which they were an incidental finding (15 cases). Of the former group, 13 were associated with additional intoxicants; two deaths were attributed solely to cathinone intoxication, both survived 10 h: a man whose postmortem blood methylone concentration was 0.71 mg/L and a woman whose postmortem blood ethylone concentration was 1.7 mg/L. In the latter category, there were several individuals who had higher concentrations of cathinones than the above two, the highest being a blood methylone of 4.8 mg/L. Based upon our data and the literature presented, lethal concentrations of cathinones cannot be established.

Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy.

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

Emerging role for autophagy in the removal of aggresomes in Schwann cells.

The presence of protein aggregates in the nervous system is associated with various pathological conditions, yet their contribution to disease mechanisms is poorly understood. One type of aggregate, the aggresome, accumulates misfolded proteins destined for degradation by the ubiquitin-proteasome pathway. Peripheral myelin protein 22 (PMP22) is a short-lived Schwann cell (SC) protein that forms aggresomes when the proteasome is inhibited or the protein is overexpressed. Duplication, deletion, or point mutations in PMP22 are associated with a host of demyelinating peripheral neuropathies, suggesting that, for normal SC cell function, the levels of PMP22 must be tightly regulated. Therefore, we speculate that mutant, misfolded PMP22 might overload the proteasome and promote aggresome formation. To test this, sciatic nerves of Trembler J (TrJ) neuropathy mice carrying a leucine-to-proline mutation in PMP22 were studied. In TrJ neuropathy nerves, PMP22 has an extended half-life and forms aggresome-like structures that are surrounded by molecular chaperones and lysosomes. On the basis of these characteristics, we hypothesized that PMP22 aggresomes are transitory, linking the proteasomal and lysosomal protein degradation pathways. Here we show that Schwann cells have the ability to eliminate aggresomes by a mechanism that is enhanced when autophagy is activated and is primarily prevented when autophagy is inhibited. This mechanism of aggresome clearance is not unique to peripheral glia, because L fibroblasts were also capable of removing aggresomes. Our results provide evidence for the involvement of the proteasome pathway in TrJ neuropathy and for the role of autophagy in the clearance of aggresomes.