Effect of insulin and an erythropoietin-derived peptide (ARA290) on established neuritic dystrophy and neuronopathy in Akita (Ins2 Akita) diabetic mouse sympathetic ganglia.

The Akita mouse is a robust model of diabetic autonomic neuropathy which develops severe diabetes following beta cell death, which occurs reproducibly at 3-4 weeks of age, and maintains the diabetic state without therapy for as long as 11 additional months. Neuritic dystrophy and neuronopathy involving prevertebral sympathetic superior mesenteric and celiac ganglia begin to develop within the first two months of onset of diabetes and are progressive with time. We have examined the effect of insulin implants resulting in normoglycemia and injections of ARA290, a small erythropoietin peptide which has no effect on glycemic parameters, on the reversal of established neuritic dystrophy and neuronopathy. We have found that 4 weeks of insulin therapy beginning at 2 months of diabetes resulted in normalization of blood glucose, body weight and HbA1c. Insulin therapy successfully reversed established neuritic dystrophy and neuronopathy to control levels. Numbers of sympathetic neurons were not significantly changed in either 3 month diabetic or insulin-treated Akita mice. Treatment with ARA290 for 7 weeks beginning at 4 months of diabetes did not result in altered metabolic severity of diabetes as measured by blood glucose, body weight or HbA1c levels. ARA290 treatment was able to decrease neuritic dystrophy by 55-74% compared to untreated diabetics or in comparison to a separate group of diabetic animals representing the 4 month treatment onset point. Surprisingly, there was no effect of ARA290 on ganglionic neuron number or ongoing neuronopathy (pale/degenerating neurons) in diabetic Akita mice during this time period. The development of neuroprotective EPO-like peptides may provide a possible future therapy for this debilitating complication of diabetes; however, it appears that discrete elements may be differentially targeted by the diabetic state and may require selective therapy.

Genetic ablation of calcium-independent phospholipase A2{gamma} leads to alterations in hippocampal cardiolipin content and molecular species distribution, mitochondrial degeneration, autophagy, and cognitive dysfunction.

Genetic ablation of calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) results in profound alterations in hippocampal phospholipid metabolism and mitochondrial phospholipid homeostasis resulting in enlarged and degenerating mitochondria leading to autophagy and cognitive dysfunction. Shotgun lipidomics demonstrated multiple alterations in hippocampal lipid metabolism in iPLA(2)gamma(-/-) mice including: 1) a markedly elevated hippocampal cardiolipin content with an altered molecular species composition characterized by a shift to shorter chain length molecular species; 2) alterations in both choline and ethanolamine glycerophospholipids, including a decreased plasmenylethanolamine content; 3) increased oxidized phosphatidylethanolamine molecular species; and 4) an increased content of ceramides. Electron microscopic examination demonstrated the presence of enlarged heteromorphic lamellar structures undergoing degeneration accompanied by the presence of ubiquitin positive spheroid inclusion bodies. Purification of these enlarged heteromorphic lamellar structures by buoyant density centrifugation and subsequent SDS-PAGE and proteomics identified them as degenerating mitochondria. Collectively, these results identify the obligatory role of iPLA(2)gamma in neuronal mitochondrial lipid metabolism and membrane structure demonstrating that iPLA(2)gamma loss of function results in a mitochondrial neurodegenerative disorder characterized by degenerating mitochondria, autophagy, and cognitive dysfunction.

Autophagy regulates pancreatic beta cell death in response to Pdx1 deficiency and nutrient deprivation.

There are three types of cell death; apoptosis, necrosis, and autophagy. The possibility that activation of the macroautophagy (autophagy) pathway may increase beta cell death is addressed in this study. Increased autophagy was present in pancreatic islets from Pdx1(+/-) mice with reduced insulin secretion and beta cell mass. Pdx1 expression was reduced in mouse insulinoma 6 (MIN6) cells by delivering small hairpin RNAs using a lentiviral vector. The MIN6 cells died after 7 days of Pdx1 deficiency, and autophagy was evident prior to the onset of cell death. Inhibition of autophagy prolonged cell survival and delayed cell death. Nutrient deprivation increased autophagy in MIN6 cells and mouse and human islets after starvation. Autophagy inhibition partly prevented amino acid starvation-induced MIN6 cell death. The in vivo effects of reduced autophagy were studied by crossing Pdx1(+/-) mice to Becn1(+/-) mice. After 1 week on a high fat diet, 4-week-old Pdx1(+/-) Becn1(+/-) mice showed normal glucose tolerance, preserved beta cell function, and increased beta cell mass compared with Pdx1(+/-) mice. This protective effect of reduced autophagy had worn off after 7 weeks on a high fat diet. Increased autophagy contributes to pancreatic beta cell death in Pdx1 deficiency and following nutrient deprivation. The role of autophagy should be considered in studies of pancreatic beta cell death and diabetes and as a target for novel therapeutic intervention.

Neuritic dystrophy and neuronopathy in Akita (Ins2(Akita)) diabetic mouse sympathetic ganglia.

Diabetic autonomic neuropathy is a debilitating, poorly studied complication of diabetes. Our previous studies of non-obese diabetic (NOD) and related mouse models identified rapidly developing, dramatic pathology in prevertebral sympathetic ganglia; however, once diabetic, the mice did not survive for extended periods needed to examine the ability of therapeutic agents to correct established neuropathy. In the current manuscript we show that the Akita (Ins2(Akita)) mouse is a robust model of diabetic sympathetic autonomic neuropathy with unambiguous, spontaneous, rapidly-developing neuropathology which corresponds closely to the characteristic pathology of other rodent models and man. Akita mice diabetic for 2, 4 or 8 months of diabetes progressively developed markedly swollen axons and dendrites ("neuritic dystrophy") in the prevertebral superior mesenteric (SMG) and celiac ganglia (CG). Comparable changes failed to develop in the superior cervical ganglia (SCG) of the Akita mouse or in any ganglia of non-diabetic mice. Morphometric studies demonstrate an overall increase in presynaptic axon terminal cross sectional area, including those without any ultrastructural features of dystrophy. Neurons in Akita mouse prevertebral sympathetic ganglia show an unusual perikaryal alteration characterized by the accumulation of membranous aggregates and minute mitochondria and loss of rough endoplasmic reticulum. These changes result in the loss of a third of neurons in the CG over the course of 8 months of diabetes. The extended survival of diabetic mice and robust pathologic findings provide a clinically relevant paradigm that will facilitate the analysis of novel therapeutic agents on the reversal of autonomic neuropathy.

Synaptic ultrastructural alterations anticipate the development of neuroaxonal dystrophy in sympathetic ganglia of aged and diabetic mice.

Neuroaxonal dystrophy, a distinctive axonopathy characterized by marked enlargement of distal axons, is the hallmark pathologic alteration in aged and diabetic human prevertebral sympathetic ganglia and in corresponding rodent models. Neuroaxonal dystrophy is thought to represent the abnormal outcome of cycles of synaptic degeneration and regeneration; a systematic study of identified axon terminals in aged and diabetic prevertebral ganglia, however, has not previously been performed. We examined the initial changes that develop in presynaptic and postsynaptic elements in sympathetic ganglia of aged and diabetic mice and found numerous synaptic changes involving both presynaptic and postsynaptic elements. Early alterations in presynaptic axon terminal size, vesicle content, and morphology culminate in the development of anastomosing membranous tubulovesicular aggregates, accumulation of autophagosomes, and amorphous debris that form a continuum with progressively larger classically dystrophic swellings. Dendritic changes consist of the development of swellings composed of delicate tubulovesicular elements and mitochondriopathy characterized by increased numbers of small mitochondria and, exclusively in aged ganglia, megamitochondria. These results support the hypothesis that neuroaxonal dystrophy results from progressive changes in presynaptic axon terminals that likely involve membrane dynamics and which are accompanied by distinctive changes in postsynaptic dendritic elements.

Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens.

The physiologic importance of autophagy proteins for control of mammalian bacterial and parasitic infection in vivo is unknown. Using mice with granulocyte- and macrophage-specific deletion of the essential autophagy protein Atg5, we show that Atg5 is required for in vivo resistance to the intracellular pathogens Listeria monocytogenes and Toxoplasma gondii. In primary macrophages, Atg5 was required for interferongamma (IFN-gamma)/LPS-induced damage to the T. gondii parasitophorous vacuole membrane and parasite clearance. While we did not detect classical hallmarks of autophagy, such as autophagosomes enveloping T. gondii, Atg5 was required for recruitment of IFN-gamma-inducible p47 GTPase IIGP1 (Irga6) to the vacuole membrane, an event that mediates IFN-gamma-mediated clearance of T. gondii. This work shows that Atg5 expression in phagocytic cells is essential for cellular immunity to intracellular pathogens in vivo, and that an autophagy protein can participate in immunity and intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking.

Erythropoietin and its carbamylated derivative prevent the development of experimental diabetic autonomic neuropathy in STZ-induced diabetic NOD-SCID mice.

Autonomic neuropathy is a significant diabetic complication resulting in increased morbidity and mortality. Studies of autopsied diabetic patients and several rodent models demonstrate that the neuropathologic hallmark of diabetic sympathetic autonomic neuropathy in prevertebral ganglia is the occurrence of synaptic pathology resulting in distinctive dystrophic neurites ("neuritic dystrophy"). Our prior studies show that neuritic dystrophy is reversed by exogenous IGF-I administration without altering the metabolic severity of diabetes, i.e. functioning as a neurotrophic substance. The description of erythropoietin (EPO) synergy with IGF-I function and the recent discovery of EPO's multifaceted neuroprotective role suggested it might substitute for IGF-I in treatment of diabetic autonomic neuropathy. Our current studies demonstrate EPO receptor (EPO-R) mRNA in a cDNA set prepared from NGF-maintained rat sympathetic neuron cultures which decreased with NGF deprivation, a result which demonstrates clearly that sympathetic neurons express EPO-R, a result confirmed by immunohistochemistry. Treatment of STZ-diabetic NOD-SCID mice have demonstrated a dramatic preventative effect of EPO and carbamylated EPO (CEPO, which is neuroprotective but not hematopoietic) on the development of neuritic dystrophy. Neither EPO nor CEPO had a demonstrable effect on the metabolic severity of diabetes. Our results coupled with reported salutary effects of EPO on postural hypotension in a few clinical studies of EPO-treated anemic diabetic and non-diabetic patients may reflect a primary neurotrophic effect of EPO on the sympathetic autonomic nervous system, rather than a primary hematopoietic effect. These findings may represent a major clinical advance since EPO has been widely and safely used in anemic patients due to a variety of clinical conditions.

Distinct pathways regulate expression of cardiac electrical and mechanical junction proteins in response to stretch.

To define mechanisms regulating expression of cell-cell junction proteins, we have developed an in vitro system in which neonatal rat ventricular myocytes were subjected to pulsatile stretch. Previously, we showed that expression of the gap junction protein, connexin (Cx) 43, is increased by approximately 2-fold after 1 hour of stretch, and this response is mediated by stretch-induced secretion of vascular endothelial growth factor (VEGF). Here, we report that the mechanical junction proteins plakoglobin, desmoplakin, and N-cadherin are also upregulated by pulsatile stretch but by a mechanism independent of VEGF or other secreted chemical signals. Stretch-induced upregulation of mechanical junction proteins was blocked by anti-beta1 and anti-beta3 integrin antibodies. Transfection of cells with adenovirus expressing GFP-FRNK, a dominant-negative inhibitor of focal adhesion kinase (FAK)-dependent signaling, blocked stretch-induced upregulation of Cx43 and mechanical junction proteins but did not block the ability of exogenous VEGF to upregulate Cx43 expression. Conditioned medium removed from uninfected cells after stretch increased Cx43 expression when added to nonstretched cells, and this effect was blocked by anti-VEGF antibodies, but stretch-conditioned medium from GFP-FRNK cells had no effect on Cx43 expression. The src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine blocked stretch-induced upregulation of mechanical junction proteins but not Cx43. Thus, stretch upregulates expression of both electrical and mechanical junction proteins via integrin-dependent activation of FAK. Stretch-induced upregulation of Cx43 expression is mediated by FAK-dependent secretion of VEGF. In contrast, stretch-induced upregulation of adhesion junction proteins involves intracellular mechanotransduction pathways initiated via integrin signaling and acting downstream of src kinase.

Remodeling of gap junctions and slow conduction in a mouse model of desmin-related cardiomyopathy.

OBJECTIVE: We studied a transgenic mouse model of human desmin-related cardiomyopathy with cardiac-specific expression of a 7-amino acid deletion mutation in desmin (D7-des) to test the hypothesis that impaired linkage between desmin and desmosomes alters expression and function of the electrical coupling protein, connexin43 (Cx43). METHODS: Expression of Cx43 and selected mechanical junctions proteins was characterized in left ventrices of D7-des and control mice by quantitative confocal microscopy and immunoblotting. Remodeling of gap junctions was also analyzed by electron microscopic morphometry. The electrophysiological phentoype of D7-des mice was characterized by electrocardiography and optical mapping of transmembrane voltage. RESULTS: Cx43 signal at intercalated disks was decreased by approximately 3-fold in D7-des ventricular tissue due to reductions in both gap junction number and size. Immunoreactive signal at cell-cell junctions was also reduced significantly for adhesion molecules and linker proteins of desmosomes and fascia adherens junctions. Electron microscopy showed decreased gap junction remodeling. However, immunoblotting showed that the total tissue content of Cx43 and mechanical junction proteins was not reduced, suggesting that diminished signal at cell-cell junctions was not due to insufficient protein expression, but to failure of these proteins to assemble properly within electrical and mechanical junctions. Remodeling of gap junctions in D7-des mice led to slowing of ventricular conduction as demonstrated by optical electrophysiological mapping. CONCLUSIONS: These results illustrate how a defect in a protein conventionally thought to fulfill a mechanical function in the heart can also lead to electrophysiological alterations that may contribute to arrhythmogenesis.

Matrix-protein-specific regulation of Cx43 expression in cardiac myocytes subjected to mechanical load.

To elucidate mechanisms responsible for mechanotransduction in the heart and define the effects of remodeling of the extracellular matrix, we cultured neonatal rat ventricular myocytes on native type I collagen, fibronectin, or denatured collagen and subjected them to uniaxial, pulsatile stretch. Changes in expression of the cardiac gap junction protein, Cx43, were measured by confocal microscopy and immunoblotting. Cells grown on fibronectin or denatured collagen exhibited significantly greater Cx43 expression than cells grown on native collagen. Stretch induced a approximately 2-fold increase in Cx43 expression in cells grown on native collagen but no increase in cells grown on fibronectin or denatured collagen. Incubation of cells on native collagen with a peptide containing the arginine-glycine-aspartate (RGD) motif upregulated Cx43 expression equivalent to that induced by stretch. Nonselective activation of integrin signaling with MnCl2 also upregulated Cx43 expression in cells grown on native collagen. This effect was blocked completely by pretreatment with anti-beta1 integrin antibody but not by anti-beta3 integrin antibody. Stretch led to a marked increase in beta1 integrin immunofluorescent signal in cells grown on native collagen but not in cells grown on fibronectin or denatured collagen. Stretch-induced upregulation of Cx43 was also blocked by anti-beta1 integrin antibody. Thus, matrix protein-myocyte interactions regulate Cx43 expression via beta1 integrin signaling initiated by mechanical stimulation in cells grown on native type I collagen, or by RGD-integrin signaling independent of mechanical stress in cells grown on fibronectin or denatured collagen. Changes in the composition of the extracellular matrix may affect electrical coupling in cardiac myocytes.

Transmural distribution of connexins in rodent hearts.

INTRODUCTION: Electrophysiologic heterogeneity across the ventricular wall is a result of differential transmural expression of various ion channel proteins that underlie the different action potential waveforms observed in epicardial, midmyocardial, and endocardial regions. Cardiac connexins mediate cell-to-cell communication, are critical for normal impulse propagation, and play a role in electrophysiologic remodeling in disease states. However, little is known about the transmural distribution of cardiac gap junction proteins. METHODS AND RESULTS: Connexin expression in epicardium, midmyocardium, and endocardium was assessed immunohistochemically in mouse and rat hearts. The total connexin protein content within different ventricular regions was measured by immunoblotting. Connexin43 is twice as abundant in midmyocardium and endocardium compared with epicardium in the mouse but not in the rat. Connexin45 is expressed equally across the left ventricular wall. CONCLUSION: Epicardial myocytes express significantly less Cx43 and therefore may be less well coupled than midmyocardial and endocardial myocytes. A transmural gradient of connexin43 expression across the left ventricular free wall likely results in differences in the stoichiometry of connexins expressed in different regions of the heart.

Redistribution of connexin45 in gap junctions of connexin43-deficient hearts.

OBJECTIVE: Adult ventricular myocytes express two gap junction channel proteins: connexin43 (Cx43) and connexin45 (Cx45). Cx43-deficient mice exhibit slow ventricular epicardial conduction, suggesting that Cx43 plays an important role in intercellular coupling in the ventricle. Cx45 is much less abundant than Cx43 in working ventricular myocytes. Its role in ventricular conduction has not been defined, nor is it known whether expression or distribution of Cx45 is altered in Cx43-deficient mice. The present study was undertaken to determine (1) whether expression of Cx45 is upregulated and (2) whether gap junction structure and distribution are altered in Cx43-deficient mice. METHODS: Ventricular tissue from neonatal Cx43(+/+), Cx43(+/-) and Cx43(-/-) and adult Cx43(+/+) and Cx43(+/-) mice was analyzed by immunoblotting and confocal immunofluorescence microscopy. RESULTS: Total Cx45 protein abundance measured by immunoblotting was not different in Cx43-deficient or null hearts compared to wild-type control hearts. However, the amount and distribution of Cx45 immunoreactive signal measured by quantitative confocal analysis were markedly reduced in both Cx43(+/-) and Cx43(-/-) hearts. CONCLUSION: Although the total content of Cx45 is not upregulated in Cx43-deficient hearts, the localization of Cx45 to cardiac gap junctions depends on the expression level of Cx43 and is dramatically altered in mice that express no Cx43.