Sera from Patients with Type 2 Diabetes and Neuropathy Induce ...

[Autophagy 1:3, 163-170; October/November/December 2005]; ©2005 Landes Bioscience

Sera from Patients with Type 2 Diabetes and Neuropathy Induce Autophagy and Colocalization with Mitochondria in SY5Y Cells Research Paper

ABSTRACT The etiology of diabetic neuropathy is multifactorial and not fully elucidated, although oxidative stress and mitochondrial dysfunction are major factors. We reported previously that complement-inactivated sera from type 2 diabetic patients with neuropathy induce apoptosis in cultured neuronal cells, possibly through an autoimmune immunoglobulinmediated pathway. Recent evidence supports an emerging role for autophagy in a variety of diseases. Here we report that exposure of human neuroblastoma SH-SY5Y cells to sera from type 2 diabetic patients with neuropathy is associated with increased levels of autophagosomes that is likely mediated by increased titers of IgM or IgG autoimmune immunoglobulins. The increased presence of macroautophagic vesicles was monitored using a specific immunohistochemical marker for autophagosomes, anti-LC3-II immunoreactivity, as well as the immunohistochemical signal for beclin-1, and was associated with increased co-localization with mitochondria in the cells exposed to diabetic neuropathic sera. We also report that dorsal root ganglia removed from streptozotocin-induced diabetic rats exhibit increased levels of autophagosomes and co-localization with mitochondria in neuronal soma, concurrent with enhanced binding of IgG and IgM autoimmune immunoglobulins. To our knowledge, this is the first evidence that the presence of autophagosomes is increased by a serum factor, likely autoantibody(ies) in a pathological condition. Stimulation of autophagy by an autoantibody-mediated pathway can provide a critical link between the immune system and the loss of function and eventual demise of neuronal tissue in type 2 diabetes.

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Sciences Institute; Department of Molecular; Cellular and Developmental Biology and Department of Biological Chemistry; University of Michigan; Ann Arbor, Michigan USA

INTRODUCTION

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*Correspondence to: John W. Wiley; University of Michigan GCRC, A7007 UH; 1500 East Medical Center Drive; Ann Arbor, Michigan 48109-0108 USA; Tel.: 734.936.8080; Fax: 734.936.4024; Email: [email protected]

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3Deptartment Cell Genetics; National Institute Genetics; Shizuoka-ken, Japan

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2Deptartment Cell Biology; National Institute Basic Biology; Okazaki, Japan

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1Department of Internal Medicine; University of Michigan; Ann Arbor, Michigan USA

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Roberto Towns1 Yukiko Kabeya2 Tamotsu Yoshimori3 Chunfang Guo1 Yu Shangguan1 Shuangsong Hong1 Mariana Kaplan1 Daniel J. Klionsky4 John W. Wiley1,*

Received 06/20/05; Accepted 08/02/05

Diabetic neuropathy is the most common peripheral neuropathy in the developed world, affecting 30–60% of diabetic patients. The etiology of this disorder is multifactorial and successful treatments have proven elusive. Recent studies have demonstrated that even in the presence of sustained euglycemia, a subset of diabetic patients develop neuropathy.1 In addition to metabolic and endocrine abnormalities, recent studies support the presence of autoimmune immunoglobulin in the sera of patients with type 1 and type 2 diabetes that induce apoptosis in cultured neurons.2-4 These autoimmune antibodies are present in complement-inactivated sera from humans5 and rat models6 and suggest a role for autoantibodies in the pathophysiology of diabetic neuropathy. It is noteworthy that the level of apoptosis observed in primary afferent nerves in situ (~1–4%) is consistent with the modest loss in neurons reported in peripheral nerve tissues in diabetes,7-9 making it difficult to account for the more robust deficits in peripheral nerve function, e.g., slowing in nerve conduction velocity, altered sensation and autonomic nervous system dysfunction observed concurrently with apoptosis. This suggests that other stress-related pathways are activated in a larger proportion of neurons compared to the small percentage of cells exhibiting apoptosis, and are likely involved in the early response of neurons to the inimical conditions occurring in type 2 diabetes. Elucidation of these events may reveal novel mechanisms of how neurons respond in diabetes and suggest new therapeutic targets to ameliorate the degree of dysfunction and eventual neuronal loss. A rapidly growing body of research suggests that macroautophagy (autophagy) plays a pivotal role in cellular response to inimical conditions.10,11 Autophagy is a ubiquitous adaptive mechanism that allows cells to survive stressful conditions such as nutrient depletion and oxidative stress and appears to play an important role in the pathophysiology of neurodegenerative, cardiovascular, and muscular diseases, and some malignancies.12-14 Autophagy is a highly conserved pathway in eukaryotic organisms. In mammals, it involves the functional homologues of at least 16 gene products, studied comprehensively in yeast.15

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Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=2068

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ACKNOWLEDGEMENTS

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autophagy, diabetic neuropathy, autoimmune immunoglobulins, mitochondria, DRG neurons, SH-SY5Y neuroblastoma cells

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This work was supported by the following grants from the National Institutes of Health (to J.W.W.): R01-056997, R01-052387 and M01-RR00042.

www.landesbioscience.com

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Autoantibody Activation of Autophagy in Diabetic Neuropathy

Among these products is the specific marker of autophagic vacuoles/ autophagosomes, LC3, a member of the microtubule-associated light chain family, a mammalian homologue of yeast Atg8.16 LC3 undergoes enzymatic cleavage and lipidation to generate the 16 KDa LC3-II form that is incorporated into autophagosomes,17 and whose immunohistochemical (IHC) detection exhibits a punctate18 or ringlike pattern19 indicative of completed autophagosomes. Therefore, the detection of LC3-II by immunofluoresence or immunoblot, provides a specific marker for the assessment of macroautophagic activity, and allows the evaluation of the co-localization of LC3 with other events involved in cellular responses to stress.20 The engulfment of organelles is a hallmark of autophagy.21 For example, sequestration of mitochondria in autophagosomes increases when cells and organisms are exposed to stressful or pathophysiological conditions.22,23 However, the physiological and pathophysiological significance of mitochondrial engulfment is not thoroughly understood. Mitochondrial biogenesis is upregulated under conditions of enhanced oxidative stress,24 the latter being a major feature of the cellular diabetic milieu.25 Therefore, it is possible that increased mitochondrial sequestration in autophagosomes is a cytoprotective response to increased levels of mediators of oxidative injury in diabetes. In this report we demonstrate that the IHC label of LC3, accompanied by an increase in LC3-II immunoblot signal and co-localization with mitochondria, is likely induced as part of an autoimmune response to immunoglobulins that are present in elevated titers in the sera of diabetic patients with neuropathy.

MATERIALS AND METHODS

Patient information. Prior approval for these studies was obtained from the University of Michigan Institutional Review Board. After informed consent was obtained, sera were collected from patients with type 2 diabetes with and without documented neuropathy, and age and gender-matched healthy adult controls. Patients were recruited from within the University of Michigan Health System clinics that specialize in diabetes care and through the use of local advertisements. Data on the sex, weight, hemoglobin A1C (HbA1C), serum glucose, serum creatinine, duration of diabetes and neuropathy are presented in Table 1. None of the patients had evidence of any other autoimmune diseases. Control patients were free of diabetes, autoimmune disease, and neuropathy. All control studies were performed on the same day as with diabetic patients. The control and diabetic samples were age matched and both were stored at 70°C for the same duration of time. As a positive control we examined the effect of sera obtained from patients with active Systemic Lupus Erythematosus (SLE) on activation of autophagy. SLE is generally accepted as a disease involving autoimmune mechanisms.26 Disease activity was assessed by the Systemic Lupus Erythematosus Disease activity index (SLEDAI).27 We accepted a SLEDAI of 2 or more to consider a patient as having active SLE. Demographics and characteristics of study patients. Clinical neuropathy was defined by established criteria.28 Briefly, neuropathy was assessed based on an abnormal neurological examination that was consistent with the presence of peripheral sensorimotor neuropathy plus either abnormal nerve conduction in at least two peripheral nerves or unequivocally abnormal autonomic nerve testing. An abnormal sensorimotor response was determined by symptoms of numbness, burning, pain or cramps in legs or feet, and signs of abnormal sensation (light touch, pain, and vibration), muscle strength, and tendon reflexes in the extremities. Nerve conduction velocities were performed on the nondominant side with the lower limb maintained at 32°C and the upper limb at 33°C. Sural, median, and ulnar sensory-evoked potential amplitudes, and distal and peak latencies were evaluated. The amplitudes of the compound muscle action potentials for the peroneal and median motor nerves and their respective distal latencies and conduction velocities were performed. A nerve was considered abnormal if any attribute

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Table 1

Demographics and characteristics of study participants

Patients (n) Age (yr) Duration of diabetes (yr) Duration of neuropathy (yr) HbA1c (%) Sex (% female) Weight (lbs) Serum creatinine (mg/dl)

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DN-

12

6

55 ± 2.3

52 ± 2.8

15.8 ± 2.6

2.1 ± 0.3

8.6 ± 1.7

N/A

8.4 ± 0.4

8.1 ± 0.3

30

20

196 ± 2.7

189 ± 2.5

1.2 ± 0.2

1.1 ± 0.1

Neuropathy was defined by the presence of delayed nerve conduction velocity by electromyogram, and/or autonomic neuropathy (refer to Materials and Methods for additional information). N/A, not applicable. All values are means ± SEM. Adult control patients showed no evidence of diabetes, autoimmune disease, or neuropathy. There were no significant differences in age, sex, weight, HbA1C or creatinine in DN+ compared to DN- patients. The duration of diabetes was significantly lower in DN- patients compared to DN+. p < 0.05 (refer to ref. 5 for additional information).

(amplitude, distal latency, or conduction velocity) was not within the normal limits, defined as values between the first and 99th percentiles. When two or more nerves were abnormal, nerve conduction was considered abnormal. Autonomic function testing. The resting heart rate was calculated after a period of 20 min supine rest. The heart rate variability response to six deep breaths/min (5 s in and 5 s out) was recorded for 1 min on a continuous electrocardiogram trace. The maximum and minimum R-R intervals during each breathing cycle were measured and converted to beats/min, and a mean value was calculated for the six measured cycles. The heart rate response to the Valsalva maneuver (expiration against a pressure of 40 mm Hg for a period of 15 s) was performed three times, and a mean value was calculated for the ratio of the longest R-R interval after the maneuver to the shortest R-R interval during the maneuver. The patient then rested supine for 20 min. After a mean supine systolic blood pressure (sBP) was measured, the patient stood erect, and the sBP was recorded immediately and at 1 min intervals, thereafter, for a further 5 min period. The lowest standing sBP was recorded, and the sBP fall calculated. If two of four tests were outside published normal values, the patient was considered to have abnormal autonomic function. Animal model: streptozotocin-induced, hypoinsulinemic diabetic rat. Two-month-old male Sprague-Dawley rats (Charles River, Wilmington, MA) were housed at the Unit for Laboratory Animal Medicine of the University of Michigan. Animals were randomized into diabetic and healthy controls. Animals were fasted for 12 h before an intraperitoneal injection of 45 mg/kg streptozotocin (Sigma, St. Louis, MO) which caused 80% of animals to become hyperglycemic within 1 week of injection, with blood glucose between 350 and 500 mg/dl. Our previous studies with this model have shown that after 4–8 weeks of diabetes the rats exhibit a variety of functional derangements, similar to those observed in human patients with diabetic neuropathy including slowing of nerve conduction velocity, increased calcium influx, impaired inhibitory G-protein function, impaired mitochondrial function and activation of the apoptosis cascade in acutely dissociated DRG neurons.5-8 Animals were narcotized through carbon dioxide inhalation and immediately perfused with 4% buffered paraformaldehyde for 20 min via a cardiac catheter. Lower thoracic and lumbar DRGs, were subsequently removed and processed for immunohistochemistry. Control animals were sham-injected with vehicle. Neuroblastoma cell culture. The human neuroblastoma cell line SH-SY5Y (ATCC, Manassas, VA) was maintained in 150 cm2 Corning T-150 flasks in a 1:1 mixture of DMEM and Ham’s F-12 containing 15% FBS, penicillin (100 IU/ml), streptomycin (100 µg/ml), 2 mM L-glutamine, and 15-mM Hepes buffer at 37°C with a 10% CO2 atmosphere. Neuronal cells were detached using Trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA) for 2 min

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Figure1. Detection of enhanced autophagy and its co-localization with immunoglobulins in DRG neurons of streptozotocin-diabetic rats compared to normal controls. (A) The IHC signals for LC3, IgG and co-localization of LC3 and IgG in DRG neurons of normal and diabetic rats were examined as described in Materials and Methods. (B) IHC detection of signal for LC3, IgM and co-localization of LC3 and IgM in DRG neurons of normal and diabetic rats. LC3 signals were increased in cells from the diabetic rats, and demonstrated coincidence with those cells that show higher levels of binding to endogenous IgG and IgM. (C) Representative immunoblot showing the relative density of the signal for LC3-I and LC3-II in DRG neurons from a control and diabetic rat. Equal amounts (20 µg) of total cellular protein were loaded per lane. The tubulin band is shown to assess gel lane loading. (D) Histogram of the relative densities for the LC3-II signal from immunoblot determinations with DRG homogenates from control and diabetic rats. The normal control was set to 100%. LC3-II levels were increased in DRGs from diabetic rats. Scale bar, 50 µm. *p < 0.05


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at 37°C and replated for the experiments at 106 cells/ 150 cm2 flask. Passage number never exceeded thirty. Nutrient deprivation (starvation) protocol. Human neuroblastoma SH-SY5Y cells were grown on a cover slip in a Petri dish at 37°C for 24 h. For the experiments involving nutrient (protein-carbohydrate) deprivation, cells were incubated in the presence and absence of 3methyladenine (3-MA; 10 mM for 1 hour at 37°C), an inhibitor of autophagy29 then the medium was replaced twice, every 2.5 hours with EBSS (Earle’s Balanced Salt Solution) buffer in the absence and presence of 10 mM 3-MA at 37°C. For non-starvation experiments, cells were incubated in the presence and absence of 10 mM 3-MA following the same sequence of incubations and media replenishments, without changes in the DMEM/ F12 culture medium. All treatment groups were subsequently fixed in 4% paraformaldehyde for 15 min. In the experiments where monodansylcadaverine (MDC)30 was used to label vacuoles, starved and non-starved cells were exposed to media or buffers containing 0.05 mM MDC for 15 min at 37°C prior to fixation. Immunofluorescence assays. Cells were grown on cover slips in a Petri dish (35 x 10 mm) or 4 well cell culture chambers for 24 h at 37°C, and fixed in ice-cold 4% paraformaldehyde for 15 min. The cells were then washed with PBS and blocked with 10% serum containing 0.1% triton X-100. The cells were then exposed to either the primary anti-LC3 antiserum,18 at 1:400 dilution, antibeclin-1 antibody at 1:100 dilution (Novus Biologicals, Littleton, CO) or anti-mitochondrial antibody (AMA) at 1:100 dilution (Chemicon International, Temecula, CA) and secondary antibodies (Alexa Fluor-495, goat anti-rabbit IgG, 1:400, and Alexa Fluor-488, goat anti-mouse IgG, 1:400, Molecular Probes, Eugene, OR) depending on the donor species of the primary antibody, in blocking buffer for 2 h at room temperature. The cells were subsequently washed in PBS and mounted along with 10 ml anti-fading gel (Molecular Probes, Eugene, OR). In the experiments with DRG tissue, 4 µm thick sections were cut and paraffin-embedded. Prior to immunostaining, the sections were deparaffinized with xylene and alcohol, rinsed with distilled water and hydrated in PBS. The sections were blocked with 10% serum in PBS and exposed to the primary antibody or antiserum as described above with the initial incubation in blocking buffer carried out overnight at 4°C. Incubation with secondary antibody as above was performed at room temperature 2 h. The cells were then rinsed, and mounted as described above. Western blotting. Cells were extracted using Trisbuffered saline (TBS), complete proteinase inhibitor cocktail (Roche, Mannheim, Germany), and 0.5% of Triton X-100. After SDS-PAGE (15% ready gel, Bio-Rad, Hercules, CA), proteins were transferred to PVDF membranes under 80 volts, for 2 h at 4°C. The membranes were subsequently blocked with 5% non-fat milk for 1 h at room temperature (RT). This was followed by an overnight incubation at 4°C with a 1:1000 dilution of anti-LC3 antiserum.18 The membrane was then washed in TBS-Tween and incubated with a goat anti-rabbit, HRP-linked secondary antibody (1:4000) (Santa Cruz Biotech, Santa Cruz, CA) for 2 h at RT. For the detection of IgG, the antibodies utilized were goat anti-human IgG-Fc, HRP-conjugated (1:3000) and for IgM detection, goat anti-human IgM HRP-conjugated (1:3000) (Bethyl Laboratories, INC, Montgomery, TX). The detection of

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tubulin, to assess gel loading, was conducted with a monoclonal anti-b-tubulin antibody of mouse origin (1:1000) (Sigma-Aldrich, St Louis, MO). The signals were detected by chemiluminescence using the SuperSignal West Dura/Pico Kit (Pierce, Rockford, IL). Protein bands on X-ray film were quantified by densitometric scanning (ImageQuant 5.0) and analyzed by SigmaPlot 2001. Immunoglobulin removal. Immunoglobulins were removed from human sera using either the Protein L kit, (Protein L-agarose; Sigma, St. Louis, MO) or a Protein A+G agarose kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions.

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Autophagy is enhanced in DRG neurons of streptozotocin-induced diabetic rats. Increase in the immunoblot signal of the autophagosomal marker LC3-II typically is associated with induction of autophagy, although the magnitude of the change is tissue and cell line-specific.20 To test the hypothesis that the presence of autophagic vesicles is increased in neurons under diabetic conditions, we assessed by IHC and immunoblot the expression of LC3-II and its Figure 2. Assessment of autophagy and the effect of autoimmune immunoglobulin removal in SH-SY5Y cells co-localization in dorsal root ganglion (DRG) exposed to normal (Nor) and diabetic neuropathic (DN) sera. (A) IHC detection of LC3 in SY5Y cells cells exhibiting enhanced immunoglobulin exposed to sera from healthy controls and sera from diabetic patients exhibiting neuropathy, before and after binding, in normal and streptozotocin-dia- removal of immunoglobulins by treatment of the sera with protein L beads. Exposure of sera to protein L betic rats, a validated model of diabetes, beads eliminated the increase in LC3 signal that resulted from exposure to sera from patients with diabetic with markedly reduced circulating insulin neuropathy. (B) Representative immunoblot and histogram demonstrating increased levels of LC3-II in SY5Y concentrations, in which the loss of b cells is cells exposed to sera from diabetic patients with neuropathy compared to normal sera. Treatment with protein induced by chemical treatment and not by L beads eliminated the increase in density of the LC3-II immunoblot. Untreated normal, control sera was set autoimmune mechanisms. IHC detection of as 100%. (C) Immunoblots of the signal for IgG and IgM autoimmune immunoglobulins in SY5Y cells treated LC3 was enhanced in the DRG soma of with sera from normal controls and patients with diabetic neuropathy before and after treatment of the sera diabetic rats compared to controls (Fig. 1A with protein L beads. SY5Y cells exposed to sera from diabetic patients demonstrated increased levels of and B). The LC3 signal often exhibited a anti-IgM > anti-IgG immunoreactivity. (D) Representative immunoblots demonstrating the effect of serial punctate pattern indicative of completed dilution of normal and diabetic neuropathic sera (n = 2) on the induction of LC3-II. Scale bar, 50 µm. *p < 0.05. autophagosomes. The IHC signal for LC3 co-localized in a significant proportion of Exposure of SH-SY5Y cells to sera (1:10 dilution) from DN+ patients the cells that displayed enhanced binding of endogenous autoimmune immunoglobulins of the IgG (Fig. 1A) and IgM (Fig. 1B) subtypes in the caused a significant increase in the induction of autophagy compared to agediabetic animals compared to controls. The immunoblot detection of LC3-II matched type 2 diabetic patients without neuropathy and sera from healthy, (Fig. 1C) and its densitometric scanning (Fig. 1D) showed a significant age-matched, non-diabetic controls using immunohistochemical detection increase in the presence of this autophagosomal marker in the DRG neurons of LC3 as the biomarker (Fig. 2A). Additionally, we performed incubations of the diabetic rats. These results support the hypothesis that the autophagy in which sera were pretreated with protein A+G agarose beads to remove a pathway is activated as part of a neuronal response to inimical conditions in range of immunoglobulins, except IgM. Under these conditions the effect diabetes mellitus. of DN+ sera on induction of autophagy was significantly reduced (data not Binding of autoimmune-immunoglobulins present in sera of patients shown). When sera were treated with protein L beads to remove with diabetic neuropathy is associated with increased levels of autophago- immunoglobulins, including IgM (Fig. 2B), the autophagy-inducing effect somes in SH-SY5Y cells. Because oxidative stress is a known inducer of of the DN+ sera was markedly reduced, more so than with the treatment autophagy,31 one possibility is that autophagy is stimulated in neurons in with protein A+G, as evidenced by IHC assay (Fig. 2A and data not shown) type 2 diabetes, as part of the pathophysiology of the disease. This possibility, and by densitometric scanning of immunoblots (Fig. 2C). To further charviewed together with our previous observations on the presence of autoimmune immunoglobulins in sera of Type 2 diabetic patients with neuropathy, acterize the autoimmune effect of the sera, a titer response assay was conducted. led us to examine the hypothesis that a serum factor, possibly autoimmune The dilution-dependency of the induction of LC3-II was supported by the immunoglobulins, present in the sera of patients with type 2 diabetes and observation that a serial dilution (1:10–1:250) of DN+ sera resulted in a neuropathy will induce autophagy in cultured human neuronal cells. To test progressive decrease in the density of the immunoblot LC3-II signal (Fig. 2D). for the presence of such a factor, we incubated human neuroblastoma Exposure of SH-SY5Y cells to sera of healthy non-diabetic patients induced (SH-SY5Y) cells in the presence of sera from patients exhibiting diabetic a modest increase in autophagy compared to no serum treatment, i.e., neuropathy (DN+, n = 12), sera from patients with type 2 diabetes without growth medium without supplemental serum (data not shown). This was neuropathy (DN-, n = 6) and sera from healthy, age and gender-matched similar to the effect observed in incubations with sera of short-duration (non-diabetic) donors (n = 6). The presence of diabetic neuropathy was diabetic patients that did not exhibit signs of neuropathy. These effects likely involve lower titers of autoimmune immunoglobulins present in the sera of assessed as described previously.5

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The autoimmune induction of the autophagic signal was also confirmed by experiments in which SH-SY5Y cells were exposed to complement-inactivated sera from patients with active systemic lupus erythematosus (SLE), a disease known to involve increased levels of autoimmue immunoglobulins with a significant role for IgM.32 SH-SY5Y neurons were exposed to normal sera or sera from patients with active SLE (n = 3), and then stained with MDC (Fig. 4D, representative response). Autophagy was stimulated by sera from patients with active SLE compared to sera from healthy controls. Together, the results suggest that autoimmune immunoglobulins from patients with either diabetic neuropathy or active SLE activate autophagy. The induction of autophagy in DRG neurons of diabetic rats is associated with increased co-localization of mitochondria. We extended our studies to assess whether diabetic neuropathy (DN) is associated with an increase in mitochondrial biogenesis and/or co-localization with LC3. DRG neurons were examined from normal and diabetic rats for anti-LC3 and anti-AMA IHC staining as described in Materials and Methods. Early diabetic neuropathy was associated with an increase in the detection of both antigens using IHC and increased co-localization of the two markers in DRG neurons from diabetic rats compared to controls (Fig. 5A). We also examined the IHC detection Figure 3. Induction of autophagy by diabetic neuropathic sera and inhibition by 3-methyladenine. (A) of beclin-1, a key component in the development Labeling of autophagic vacuoles with the lysomotropic dye monodansylcadaverine (MDC) in SH-SY5Y of the autophagosome15 and observed significells exposed to normal or diabetic neuropathic sera, in the absence and presence of the autophagy cant co-localization with the LC3 signal (Fig. 5B), inhibitor, 3-methyladenine (3-MA). MDC staining increased in cells exposed to serum from patients with thereby providing additional support for diabetic neuropathy. (B) Histogram of the MDC label in the experiments conducted with SH-SY5Y cells increased induction of autophagy in the DRG treated with normal or diabetic neuropathic sera, in the absence and presence of 3-MA. (C) neurons in diabetic rats. There was an increase Representative immunoblot of LC3-I and LC3-II and (D) histogram showing the induction of LC3-II after in the density of the AMA band detected by exposure of SH-SY5Y cells to normal or diabetic neuropathic sera in the absence and presence of 3-MA. immunoblot (Fig. 5C and D) in the diabetic rats, Scale Bar, 50 µm. *p < 0.05. likely reflecting an increase in mitochondrial biogenesis. Exposure of SH-SY5Y cells to sera from diabetic patients with neuropahealthy controls as evidenced by the decrease in Figure 2A–C (Nor) in the thy is associated with induction of autophagy and co-localization with LC3 signal after removal of immunoglobulins with protein L beads. Induction of autophagy by sera from diabetic patients with neuropathy mitochondria. SH-SY5Y cells were treated with normal and diabetic neurois similar to induction of autophagy by starvation. Additional experiments pathic sera and examined by the IHC and western blot assays as described were conducted in which the induction of autophagy by DN+ sera was in Materials and Methods. Cells that were exposed to diabetic neuropathic examined with monodansylcadaverine (MDC), a marker for acidified com- sera displayed increased levels of both anti-LC3 and anti-AMA immunorepartments including autophagosomes. In these studies, exposure of the cells activity (Fig. 6). The enhanced intensity and punctate appearance of the to sera from diabetic patients with neuropathy caused an increase in MDC IHC signal for LC3 in cells incubated with sera from diabetic patients, labeling (Fig. 3A and B) and LC3-II levels (Fig. 3D and E), suggesting a compared to healthy controls, also exhibited increased co-localization with higher level of autophagy. In contrast, there was no increase in either MDC the AMA label (Fig. 6A), similar to that observed in diabetic rat DRGs, staining (Fig. 3A and B) or immunoreactive LC3-II (Fig. 3C and D) when supporting increased engulfment of mitochondrial in autophagosomes. The the incubations were conducted in the presence of the autophagy inhibitor detection of an enhanced signal for AMA in cells exposed to diabetic 3-methyladenine (3-MA). The inhibition seen in the presence of 3-MA neuropathic sera using IHC were confirmed by the increase in the density suggests that the increased LC3 levels detected in response to sera from of the AMA band by immunoblot (Fig. 6B and C). Our results with the SHpatients with diabetic neuropathy is a result of increased autophagy and is SY5Y cell model, where glucose levels are similar across incubations, suggest that the increased presence of the autoimmune immunoglobulin(s) or other not due to an unrelated phenomenon. To estimate the physiological significance of the magnitude of the LC3- serum factor present in diabetic neuropathic sera is sufficient to enhance the II increase observed when SH-SY5Y were exposed to diabetic neuropathic signals for autophagosomes, as well as increased mitochondrial immunoresera, we also examined LC3-II levels following starvation, which is known to activity. induce autophagy. When SH-SY5Y cells were deprived of nutrients, there was an increase in LC3 IHC staining (Fig. 4A) and an increase in immunodetectable LC3-II (Fig. 4B and C). The starvation-induced increases were similar in magnitude to those observed in the experiments employing sera from diabetic patients with neuropathy (Fig. 3).

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To our knowledge, this is the first report providing evidence that autoimmune immunoglobulins can stimulate autophagy and mitochondrial sequestration as part of the pathophysiology of diabetic neuropathy. The experiments showing increased labeling of autophagosomes with antiLC3 immunoreactivity in DRG neurons of streptozotocindiabetic rats follow our earlier observations highlighting a potential role for autoimmunity in the induction of cellular stress6 and apoptosis in diabetic neuropathy.5 Those observations are extended in the present report by the experiments conducted with SH-SY5Y cells, exposed to normal or diabetic neuropathic sera, in media containing B C similar concentrations of glucose and other nutrients. The increased signal for anti-LC3 immunoreactivity in SY5Y cells exposed to sera from patients with diabetic neuropathy suggests that autoimmune immunoglobulins or, perhaps, other factors present in the sera can by themselves increase autophagy. The potential significance of autoimmune immunoglobulin-induced autophagy in the natural history of diabetic neuropathy remains to be fully elucidated. However, the demonstration that sera obtained from patients with active SLE also show increased MDC labeling D of vesicles, supports the hypothesis that autoimmune disorders may, in general, result in an activation of autophagy. Interestingly, SLE is known to have an important neural component33,34 and as in our results with diabetic neuropathic sera, the autoimmune effect of SLE involves immunoglobulins of the IgM subclass.32 Therefore, autophagy may play a significant role in the cellular response when neuronal tissue is stressed by increased presence of autoimmune immunoglobulins or other factors present in diabetic sera of patients exhibiting neuropathy. The experiments demonstrating that exposure of diabetic neuropathic sera to protein L agarose beads removes immunoglobulins and activates autopahgy supports the Figure 4. Starvation-induced autophagy in SH-SY5Y cells. (A) IHC label of LC3 in hypothesis that the serum factors are indeed SH-SY5Y cells in conditions of nutrient availability (nonstarved) and nutrient deprivation immunoglobulins. However, confirmation of this hypoth- (starved). (B) Representative immunoblot showing the bands for LC3-I and LC3-II and (C) esis will require isolation and re-addition of the pertinent histogram depicting the induction of LC3-II in SH-SY5Y cells in conditions of nutrient availability (nonstarved) and nutrient deprivation (starved). Starvation-induced immunoglobulins in order to fully substantiate this possi- autophagy demonstrates a similar magnitude of increase in LC3 levels as exposure to bility. These experiments are under development in our serum from patients with diabetic neuropathy. Normal control sera and non-starved laboratory. treatments were set to 100% in histograms. (D) MDC labeling was also examined in The pathophysiological significance of mitochondrial SH-SY5Y neurons exposed to sera from normal (Nor) controls or patients with active SLE co-localization in autophagosomes remains poorly under- (n = 3). A representative response is depicted demonstrating that exposure to serum from stood. For example, mitochondria are known to generate a patient with active SLE was associated with increased formation of MDC-positive reactive oxygen species (ROS) when cells are exposed to vacuoles compared to treatment with serum from a healthy control. Scale bars, 50 µm. *p < 0.05. inimical conditions associated with oxidative stress35 and as such, an upregulation in mitochondrial biogenesis and mass, which are known to occur under conditions of increased oxida- hypothesis is that the increased presence of a serum factor, likely tive stress,24 may be a significant factor in the further generation of autoimmune immunoglobulin(s), is associated ultimately with cell ROS and subsequent worsening of the cellular environment. injury. However, it is possible that at early stages the induction of Therefore, increased mitochondrial sequestration in autophagic autophagic engulfment of mitochondria in response to binding of vacuoles may be an early cytoprotective response to oxidative autoimmune immunoglobulins prevents mitochondrial-induced stress.36 The importance of oxidative stress in the cellular damage oxidative stress from causing damage to the nerve tissue. While the that occurs in diabetic neuropathy is well documented.25 We active engulfment of mitochondria under conditions of cellular stress hypothesize that autophagy is an early cytoprotective response to the is known to occur,21,23,36,37 the fate of the engulfed mitochondria is binding of autoimmue immunoglobulins or a serum factor that activate not fully understood. The targeting of sequestered mitochondria for cellular pathways associated with mitochondrial dysfunction and eventual degradation seems a logical consequence.21 However, some increased formation of ROS. If confirmed, a logical extension of this studies suggest that in neurodegenerative conditions, the maturation 168

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Figure 5. Diabetic rats demonstrate increased levels of anti-LC3 and antimitochondria (AMA) immunoreactivity that co-localize in DRG neurons. (A) IHC detection of LC3 and co-localization with the mitochondrial AMA signal in DRG neurons from control and diabetic rats. (B) IHC detection of beclin-1, LC3 and their co-localization in DRG neurons of normal, control and diabetic rats. (C) Representative immunoblot of mitochondrial (AMA) label in DRG homogenates from normal (control) diabetic rats. (D) Histogram of densitometric scannings of immunoblots of mitochondria (AMA) in DRG homogenates from control and diabetic rat DRG neurons. The normal control was set to 100%. Scale bar, 50 µm. *p < 0.05.

of autophagic vacuoles to lysosomes may also be impaired.37 Nevertheless, it is unlikely that the engulfment of injured mitochondria in autophagic vesicles would prevent further cell damage indefinitely, e.g., chronic illness will ultimately progress to cell death in the absence of a successful therapeutic intervention. Our previous reports5,7 suggest that diabetic neuropathic sera can indeed induce apoptosis in neural tissue. The current study suggests that induction of autophagy by diabetic neuropathic sera may be an important response to the inimical cellular conditions associated with neuropathy in type 2 diabetes mellitus. www.landesbioscience.com

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Figure 6. SH-SY5Y neurons treated with sera from patients with diabetic neuropathy (DN) demonstrated an increase in anti-LC3 and anti-AMA immunoreactivity, and co-localization of the two labels compared to cells treated with normal (Nor) sera. (A) Increased IHC detection for LC3 and co-localization of the anti-mitochondrial (AMA) label in SH-SY5Y cells incubated with sera from diabetic patients with neuropathy. (B) Immunohistochemical detection of the co-localization (white arrows) of the signal for LC3 and mitochondria (AMA) in SH-SY5Y cells treated with diabetic neuropathic sera. (C) Representative immunoblot and histogram depicting densitometric scannings of the AMA detection of mitochondrial proteins in homogenates from SH-SY5Y neuroblastoma cells incubated with normal sera (Nor) or diabetic neuropathic (DN) sera. The normal control was set to 100%. Scale Bar, 50 µm. *p < 0.05.

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