Publications by authors named "Stephen I Lentz"

31 Publications

Acitretin mitigates uroporphyrin-induced bone defects in congenital erythropoietic porphyria models.

Sci Rep 2021 May 5;11(1):9601. Epub 2021 May 5.

Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, 08854, USA.

Congenital erythropoietic porphyria (CEP) is a rare genetic disorder leading to accumulation of uro/coproporphyrin-I in tissues due to inhibition of uroporphyrinogen-III synthase. Clinical manifestations of CEP include bone fragility, severe photosensitivity and photomutilation. Currently there is no specific treatment for CEP, except bone marrow transplantation, and there is an unmet need for treating this orphan disease. Fluorescent porphyrins cause protein aggregation, which led us to hypothesize that uroporphyrin-I accumulation leads to protein aggregation and CEP-related bone phenotype. We developed a zebrafish model that phenocopies features of CEP. As in human patients, uroporphyrin-I accumulated in the bones of zebrafish, leading to impaired bone development. Furthermore, in an osteoblast-like cell line, uroporphyrin-I decreased mineralization, aggregated bone matrix proteins, activated endoplasmic reticulum stress and disrupted autophagy. Using high-throughput drug screening, we identified acitretin, a second-generation retinoid, and showed that it reduced uroporphyrin-I accumulation and its deleterious effects on bones. Our findings provide a new CEP experimental model and a potential repurposed therapeutic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-88668-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100164PMC
May 2021

mTORC1 and mTORC2 expression in inner retinal neurons and glial cells.

Exp Eye Res 2020 08 2;197:108131. Epub 2020 Jul 2.

Ophthalmology & Visual Sciences, United States; Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, United States; Molecular and Integrative Physiology, University of Michigan Medical School, United States. Electronic address:

The retina is one of the most metabolically active tissues, yet the processes that control retinal metabolism remains poorly understood. The mTOR complex (mTORC) that drives protein and lipid biogenesis and autophagy has been studied extensively in regards to retinal development and responses to optic nerve injury but the processes that regulate homeostasis in the adult retina have not been determined. We previously demonstrated that normal adult retina has high rates of protein synthesis compared to skeletal muscle, associated with high levels of mechanistic target of rapamycin (mTOR), a kinase that forms multi-subunit complexes that sense and integrate diverse environmental cues to control cell and tissue physiology. This study was undertaken to: 1) quantify expression of mTOR complex 1 (mTORC1)- and mTORC2-specific partner proteins in normal adult rat retina, brain and liver; and 2) to localize these components in normal human, rat, and mouse retinas. Immunoblotting and immunoprecipitation studies revealed greater expression of raptor (exclusive to mTORC1) and rictor (exclusive for mTORC2) in normal rat retina relative to liver or brain, as well as the activating mTORC components, pSIN1 and pPRAS40. By contrast, liver exhibits greater amounts of the mTORC inhibitor, DEPTOR. Immunolocalization studies for all three species showed that mTOR, raptor, and rictor, as well as most other known components of mTORC1 and mTORC2, were primarily localized in the inner retina with mTORC1 primarily in retinal ganglion cells (RGCs) and mTORC2 primarily in glial cells. In addition, phosphorylated ribosomal protein S6, a direct target of the mTORC1 substrate ribosomal protein S6 kinase beta-1 (S6K1), was readily detectable in RGCs, indicating active mTORC1 signaling, and was preserved in human donor eyes. Collectively, this study demonstrates that the inner retina expresses high levels of mTORC1 and mTORC2 and possesses active mTORC1 signaling that may provide cell- and tissue-specific regulation of homeostatic activity. These findings help to define the physiology of the inner retina, which is key for understanding the pathophysiology of optic neuropathies, glaucoma and diabetic retinopathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exer.2020.108131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484061PMC
August 2020

Actions of Rab27B-GTPase on mammalian central excitatory synaptic transmission.

Physiol Rep 2020 05;8(9):e14428

Molecular & Integrative Physiology Department, University of Michigan, Ann Arbor, MI, USA.

Members of the Rab3 gene family are considered central to membrane trafficking of synaptic vesicles at mammalian central excitatory synapses. Recent evidence, however, indicates that the Rab27B-GTPase, which is highly homologous to the Rab3 family, is also enriched on SV membranes and co-localize with Rab3A and Synaptotagmin at presynaptic terminals. While functional roles of Rab3A have been well-established, little functional information exists on the role of Rab27B in synaptic transmission. Here we report on functional effects of Rab27B at SC-CA1 and DG-MF hippocampal synapses. The data establish distinct functional actions of Rab27B and demonstrate functions of Rab27B that differ between SC-CA1 and DG-MF synapses. Rab27B knockout reduced frequency facilitation compared to wild-type (WT) controls at the DG/MF-CA3 synaptic region, while increasing facilitation at the SC-CA1 synaptic region. Remarkably, Rab27B KO resulted in a complete elimination of LTP at the MF-CA3 synapse with no effect at the SC-CA1 synapse. These actions are similar to those previously reported for Rab3A KO. Specificity of action on LTP to Rab27B was confirmed as LTP was rescued in response to lentiviral infection and expression of human Rab27B, but not to GFP, in the DG in the Rab27B KO mice. Notably, the effect of Rab27B KO on MF-CA3 LTP occurred in spite of continued expression of Rab3A in the Rab27B KO. Overall, the results provide a novel perspective in suggesting that Rab27B and Rab3A act synergistically, perhaps via sequential effector recruitment or signaling for presynaptic LTP expression in this hippocampal synaptic region.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.14814/phy2.14428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195558PMC
May 2020

Magnetization transfer ratio quantifies polyneuropathy in hereditary transthyretin amyloidosis.

Ann Clin Transl Neurol 2020 05 25;7(5):799-807. Epub 2020 Apr 25.

Amyloidosis Center Heidelberg, Heidelberg University Hospital, Heidelberg, Germany.

Objective: To quantify peripheral nerve lesions in symptomatic and asymptomatic hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PNP) by analyzing the magnetization transfer ratio (MTR) of the sciatic nerve, and to test its potential as a novel biomarker for macromolecular changes.

Methods: Twenty-five patients with symptomatic ATTRv-PNP, 30 asymptomatic carriers of the mutant transthyretin gene (mutTTR), and 20 age-/sex-matched healthy controls prospectively underwent magnetization transfer contrast imaging at 3 Tesla. Two axial three-dimensional gradient echo sequences with and without an off-resonance saturation rapid frequency pulse were conducted at the right distal thigh. Sciatic nerve regions of interest were manually drawn on 10 consecutive axial slices in the images without off-resonance saturation, and then transferred to the corresponding slices that were generated by the sequence with the off-resonance saturation pulse. Subsequently, the MTR and cross-sectional area (CSA) of the sciatic nerve were evaluated. Detailed neurologic and electrophysiologic examinations were conducted in all ATTRv-PNP patients and mutTTR-carriers.

Results: Sciatic nerve MTR and CSA reliably differentiated between ATTRv-PNP, mutTTR-carriers, and controls. MTR was lower in ATTRv-PNP (26.4 ± 0.7; P < 0.0001) and in mutTTR-carriers (32.6 ± 0.8; P = 0.0005) versus controls (39.4 ± 2.1), and was also lower in ATTRv-PNP versus mutTTR-carriers (P = 0.0009). MTR correlated negatively with the NIS-LL and positively with CMAPs and SNAPs. CSA was higher in ATTRv-PNP (34.3 ± 1.7 mm ) versus mutTTR-carriers (26.0 ± 1.1 mm ; P = 0.0005) and versus controls (20.4 ± 1.2 mm ; P < 0.0001). CSA was also higher in mutTTR-carriers versus controls.

Interpretation: MTR is a novel imaging marker that can quantify macromolecular changes in ATTRv-PNP and differentiate between symptomatic ATTRv-PNP and asymptomatic mutTTR-carriers and correlates with electrophysiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/acn3.51049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261747PMC
May 2020

Safety of Long-Term Storage and Shipping of Prestripped, Prestained, and Preloaded Descemet Membrane Endothelial Keratoplasty Tissue.

Cornea 2019 Aug;38(8):1023-1028

Department of Ophthalmology and Visual Sciences, W. K. Kellogg Eye Center, University of Michigan, Ann Arbor, MI.

Purpose: The purpose of this study was to determine the safety of long-term storage and shipping of prestripped, prestained, and preloaded Descemet membrane endothelial keratoplasty (pDMEK) grafts.

Methods: A total of 33 cadaveric corneas were prestripped, prestained, and preloaded using modified Jones tube injectors as pDMEK. The corneas were masked to groups that were prepared <9 hours (control), 48 hours, and 72 hours before unloading and analysis. The 48- and 72-hour tissues were shipped by airfreight on each day before arrival to simulate domestic and international shipping. The corneas were then stained using Calcein AM vital dye (Molecular Probes, Eugene, OR) and imaged using an inverted confocal microscope. Primary outcome measures were endothelial cell loss (ECL, %) and sustainability of staining. MetaMorph software (Molecular Devices, Downingtown, PA) was used to quantify ECL, and staining was evaluated subjectively using all-or-none rating.

Results: There was no difference in the mean ECL for the control, 48-hour, and 72-hour groups, which were 25.1% ± 8.8%, 26.4% ± 17.5%, and 19.2% ± 11.5%, respectively (P = 0.45; Kruskal-Wallis test). In all tissues of each group, no loss of staining was identified at each time point of analysis.

Conclusions: ECL in pDMEK tissue prepared 48 and 72 hours in advance and shipped using standard methods is similar to that in pDMEK tissue prepared on the same day. These findings support the safety of domestic and international shipping of pDMEK grafts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/ICO.0000000000001974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7315383PMC
August 2019

The Divergent Roles of Dietary Saturated and Monounsaturated Fatty Acids on Nerve Function in Murine Models of Obesity.

J Neurosci 2019 05 18;39(19):3770-3781. Epub 2019 Mar 18.

Departments of Neurology, and

Neuropathy is the most common complication of prediabetes and diabetes and presents as distal-to-proximal loss of peripheral nerve function in the lower extremities. Neuropathy progression and disease severity in prediabetes and diabetes correlates with dyslipidemia in man and murine models of disease. Dyslipidemia is characterized by elevated levels of circulating saturated fatty acids (SFAs) that associate with the progression of neuropathy. Increased intake of monounsaturated fatty acid (MUFA)-rich diets confers metabolic health benefits; however, the impact of fatty acid saturation in neuropathy is unknown. This study examines the differential effect of SFAs and MUFAs on the development of neuropathy and the molecular mechanisms underlying the progression of the complication. Male mice fed a high-fat diet rich in SFAs developed robust peripheral neuropathy. This neuropathy was completely reversed by switching the mice from the SFA-rich high-fat diet to a MUFA-rich high-fat diet; nerve conduction velocities and intraepidermal nerve fiber density were restored. A MUFA oleate also prevented the impairment of mitochondrial transport and protected mitochondrial membrane potential in cultured sensory neurons treated with mixtures of oleate and the SFA palmitate. Moreover, oleate also preserved intracellular ATP levels, prevented apoptosis induced by palmitate treatment, and promoted lipid droplet formation in sensory neurons, suggesting that lipid droplets protect sensory neurons from lipotoxicity. Together, these results suggest that MUFAs reverse the progression of neuropathy by protecting mitochondrial function and transport through the formation of intracellular lipid droplets in sensory neurons. There is a global epidemic of prediabetes and diabetes, disorders that represent a continuum of metabolic disturbances in lipid and glucose metabolism. In the United States, 80 million individuals have prediabetes and 30 million have diabetes. Neuropathy is the most common complication of both disorders, carries a high morbidity, and, despite its prevalence, has no treatments. We report that dietary intervention with monounsaturated fatty acids reverses the progression of neuropathy and restores nerve function in high-fat diet-fed murine models of peripheral neuropathy. Furthermore, the addition of the monounsaturated fatty acid oleate to sensory neurons cultured under diabetic conditions shows that oleate prevents impairment of mitochondrial transport and mitochondrial dysfunction through a mechanism involving formation of axonal lipid droplets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.3173-18.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510336PMC
May 2019

Chain length of saturated fatty acids regulates mitochondrial trafficking and function in sensory neurons.

J Lipid Res 2019 01 15;60(1):58-70. Epub 2018 Nov 15.

Departments of Neurology University of Michigan, Ann Arbor, MI 48109

Dyslipidemia associated with T2D leads to diabetic neuropathy, a complication characterized by sensory neuronal dysfunction and peripheral nerve damage. Sensory dorsal root ganglion (DRG) neurons are dependent on axonal mitochondrial energy production facilitated by mitochondrial transport mechanisms that distribute mitochondria throughout the axon. Because long-chain saturated FAs (SFAs) damage DRG neurons and medium-chain SFAs are reported to improve neuronal function, we evaluated the impact of SFA chain length on mitochondrial trafficking, mitochondrial function, and apoptosis. DRG neurons were exposed to SFAs with C12:0-C18:0 chain lengths and evaluated for changes in mitochondrial trafficking, mitochondrial polarization, and apoptosis. DRG neurons treated with C16:0 and C18:0 SFAs showed a significant decrease in the percentage of motile mitochondria and velocity of mitochondrial trafficking, whereas C12:0 and C14:0 SFAs had no impact on motility. Treatment with C16:0 and C18:0 SFAs exhibited mitochondrial depolarization correlating with impaired mitochondrial motility; the C12:0- and C14:0-treated neurons retained mitochondrial polarization. The reduction in mitochondrial trafficking and function in C16:0- and C18:0-treated DRG neurons correlated with apoptosis that was blocked in C12:0 and C14:0 SFA treatments. These results suggest that SFA chain length plays an important role in regulating axonal mitochondrial trafficking and function in DRG neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.M086843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314260PMC
January 2019

Corneal confocal microscopy for identification of diabetic sensorimotor polyneuropathy: a pooled multinational consortium study.

Diabetologia 2018 08 4;61(8):1856-1861. Epub 2018 Jun 4.

Centre for Endocrinology and Diabetes, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester, M13 9PL, UK.

Aims/hypothesis: Small cohort studies raise the hypothesis that corneal nerve abnormalities (including corneal nerve fibre length [CNFL]) are valid non-invasive imaging endpoints for diabetic sensorimotor polyneuropathy (DSP). We aimed to establish concurrent validity and diagnostic thresholds in a large cohort of participants with and without DSP.

Methods: Nine hundred and ninety-eight participants from five centres (516 with type 1 diabetes and 482 with type 2 diabetes) underwent CNFL quantification and clinical and electrophysiological examination. AUC and diagnostic thresholds were derived and validated in randomly selected samples using receiver operating characteristic analysis. Sensitivity analyses included latent class models to address the issue of imperfect reference standard.

Results: Type 1 and type 2 diabetes subcohorts had mean age of 42 ± 19 and 62 ± 10 years, diabetes duration 21 ± 15 and 12 ± 9 years and DSP prevalence of 31% and 53%, respectively. Derivation AUC for CNFL was 0.77 in type 1 diabetes (p < 0.001) and 0.68 in type 2 diabetes (p < 0.001) and was approximately reproduced in validation sets. The optimal threshold for automated CNFL was 12.5 mm/mm in type 1 diabetes and 12.3 mm/mm in type 2 diabetes. In the total cohort, a lower threshold value below 8.6 mm/mm to rule in DSP and an upper value of 15.3 mm/mm to rule out DSP were associated with 88% specificity and 88% sensitivity.

Conclusions/interpretation: We established the diagnostic validity and common diagnostic thresholds for CNFL in type 1 and type 2 diabetes. Further research must determine to what extent CNFL can be deployed in clinical practice and in clinical trials assessing the efficacy of disease-modifying therapies for DSP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00125-018-4653-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061173PMC
August 2018

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers.

J Vis Exp 2017 09 29(127). Epub 2017 Sep 29.

Department of Internal Medicine, University of Michigan;

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/53369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752341PMC
September 2017

Quantitative Analysis of Endothelial Cell Loss in Preloaded Descemet Membrane Endothelial Keratoplasty Grafts.

Cornea 2017 Nov;36(11):1295-1301

*Kellogg Eye Center, University of Michigan, Ann Arbor, MI;†Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD;‡Devers Eye Institute, Portland, OR;§Eversight Michigan, Ann Arbor, MI; and¶Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, MI, USA.

Purpose: Availability of preloaded Descemet membrane endothelial keratoplasty (pDMEK) tissue may increase acceptance of DMEK in surgical management of endothelial disease. The goal of this study was to determine the safety of pDMEK grafts for 24 hours before surgery by analyzing endothelial cell loss (ECL) using 2 image analysis software programs.

Methods: A total of 18 cadaveric corneas were prepared for DMEK using a standardized technique and loaded in a modified Jones tube injector. Nine of the corneas were injected into Calcein AM vital dye after 1 minute (controls), and the remaining 9 corneas were left preloaded for 24 hours before injection into vital dye for staining. The stained corneas were imaged using an inverted confocal microscope. ECL was then analyzed and quantified by 2 different graders using 2 image analysis software programs.

Results: The control DMEK tissue resulted in 22.0% ± 4.0% ECL compared with pDMEK tissue, which resulted in 19.2% ± 7.2% ECL (P = 0.31). Interobserver agreement was 0.93 for MetaMorph and 0.92 for Fiji. The average time required to process images with MetaMorph was 2 ± 1 minutes and with Fiji was 20 ± 10 minutes. Intraobserver agreement was 0.97 for MetaMorph and 0.93 for Fiji.

Conclusions: Preloading DMEK tissue is safe and may provide an alternative technique for tissue distribution and surgery for DMEK. The use of MetaMorph software for quantifying ECL is a novel and accurate imaging method with increased efficiency and reproducibility compared with the previously validated Fiji.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/ICO.0000000000001301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657275PMC
November 2017

Dyslipidemia impairs mitochondrial trafficking and function in sensory neurons.

FASEB J 2018 01 13;32(1):195-207. Epub 2017 Sep 13.

Department of Neurology, University of Michigan, Ann Arbor, Michigan, USA;

Mitochondrial trafficking plays a central role in dorsal root ganglion (DRG) neuronal cell survival and neurotransmission by transporting mitochondria from the neuronal cell body throughout the bundles of DRG axons. In type 2 diabetes (T2DM), dyslipidemia and hyperglycemia damage DRG neurons and induce mitochondrial dysfunction; however, the impact of free fatty acids and glucose on mitochondrial trafficking in DRG neurons remains unknown. To evaluate the impact of free fatty acids compared to hyperglycemia on mitochondrial transport, primary adult mouse DRG neuron cultures were treated with physiologic concentrations of palmitate and glucose and assessed for alterations in mitochondrial trafficking, mitochondrial membrane potential, and mitochondrial bioenergetics. Palmitate treatment significantly reduced the number of motile mitochondria in DRG axons, but physiologic concentrations of glucose did not impair mitochondrial trafficking dynamics. Palmitate-treated DRG neurons also exhibited a reduction in mitochondrial velocity, and impaired mitochondrial trafficking correlated with mitochondrial depolarization in palmitate-treated DRG neurons. Finally, we found differential bioenergetic effects of palmitate and glucose on resting and energetically challenged mitochondria in DRG neurons. Together, these results suggest that palmitate induces DRG neuron mitochondrial depolarization, inhibiting axonal mitochondrial trafficking and altering mitochondrial bioenergetic capacity.-Rumora, A. E., Lentz, S. I., Hinder, L. M., Jackson, S. W., Valesano, A., Levinson, G. E., Feldman, E. L. Dyslipidemia impairs mitochondrial trafficking and function in sensory neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201700206RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191072PMC
January 2018

Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

Biochem Biophys Res Commun 2016 Mar 2;471(4):610-5. Epub 2016 Feb 2.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA; Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2016.01.180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813283PMC
March 2016

A precursor-inducible zebrafish model of acute protoporphyria with hepatic protein aggregation and multiorganelle stress.

FASEB J 2016 05 2;30(5):1798-810. Epub 2016 Feb 2.

Department of Molecular and Integrative Physiology, Department of Internal Medicine, and Veterans Affairs Ann Arbor Healthcare System, Ann Arbor, Michigan, USA

Protoporphyria is a metabolic disease that causes excess production of protoporphyrin IX (PP-IX), the final biosynthetic precursor to heme. Hepatic PP-IX accumulation may lead to end-stage liver disease. We tested the hypothesis that systemic administration of porphyrin precursors to zebrafish larvae results in protoporphyrin accumulation and a reproducible nongenetic porphyria model. Retro-orbital infusion of PP-IX or the iron chelator deferoxamine mesylate (DFO), with the first committed heme precursor α-aminolevulinic acid (ALA), generates high levels of PP-IX in zebrafish larvae. Exogenously infused or endogenously produced PP-IX accumulates preferentially in the liver of zebrafish larvae and peaks 1 to 3 d after infusion. Similar to patients with protoporphyria, PP-IX is excreted through the biliary system. Porphyrin accumulation in zebrafish liver causes multiorganelle protein aggregation as determined by mass spectrometry and immunoblotting. Endoplasmic reticulum stress and induction of autophagy were noted in zebrafish larvae and corroborated in 2 mouse models of protoporphyria. Furthermore, electron microscopy of zebrafish livers from larvae administered ALA + DFO showed hepatocyte autophagosomes, nuclear membrane ruffling, and porphyrin-containing vacuoles with endoplasmic reticulum distortion. In conclusion, systemic administration of the heme precursors PP-IX or ALA + DFO into zebrafish larvae provides a new model of acute protoporphyria with consequent hepatocyte protein aggregation and proteotoxic multiorganelle alterations and stress.-Elenbaas, J. S., Maitra, D., Liu, Y., Lentz, S. I., Nelson, B., Hoenerhoff, M. J., Shavit, J. A., Omary, M. B. A precursor-inducible zebrafish model of acute protoporphyria with hepatic protein aggregation and multiorganelle stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201500111RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836371PMC
May 2016

Disrupted TSH Receptor Expression in Female Mouse Lung Fibroblasts Alters Subcellular IGF-1 Receptor Distribution.

Endocrinology 2015 Dec 21;156(12):4731-40. Epub 2015 Sep 21.

Department of Ophthalmology and Visual Sciences, Kellogg Eye Center and Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48105.

A relationship between the actions of TSH and IGF-1 was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR and IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the noncanonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunofluorescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without and with intact TSHR expression. IGF-1 treatment of TSHR(+/+) fibroblasts resulted in reduced nuclear and cytoplasmic staining for IGF-1Rα, whereas it enhanced the nuclear signal in TSHR(-/-) cells. In contrast, IGF-1 enhanced cytoplasmic IGF-1Rβ in TSHR(-/-) fibroblasts while increasing the nuclear signal in TSHR(+/+) cells. These findings indicate the intimate relationship between TSHR and IGF-1R found earlier in human orbital fibroblasts also exists in mouse lung fibroblasts. Furthermore, the presence of TSHR in these fibroblasts influenced not only the levels of IGF-1R protein but also its subcellular distribution and response to IGF-1. They suggest that the mouse might serve as a suitable model for delineating the molecular mechanisms overarching these two receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2015-1464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655214PMC
December 2015

Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion.

PLoS One 2015 7;10(5):e0125596. Epub 2015 May 7.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, United States of America; Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125596PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423933PMC
April 2016

Experimental evaluation and computational modeling of tissue damage from low-flow push-pull perfusion sampling in vivo.

J Neurosci Methods 2015 Mar 19;242:97-105. Epub 2015 Jan 19.

University of Michigan, Department of Chemistry, 930N University Ave, Ann Arbor, MI, 48109, United States. Electronic address:

Background: Neurochemical monitoring via sampling probes is valuable for deciphering neurotransmission in vivo. Microdialysis is commonly used; however, the spatial resolution is poor.

New Method: Recently push-pull perfusion at low flow rates (50nL/min) has been proposed as a method for in vivo sampling from the central nervous system. Tissue damage from such probes has not been investigated in detail. In this work, we evaluated acute tissue response to low-flow push-pull perfusion by infusing the nuclear stains Sytox Orange and Hoechst 33342 through probes implanted in the striatum for 200min, to label damaged and total cells, respectively, in situ.

Results: Using the damaged/total labeled cell ratio as a measure of tissue damage, we found that 33±8% were damaged within the dye region around a microdialysis probe. We found that low-flow push-pull perfusion probes damaged 24±4% of cells in the sampling area. Flow had no effect on the number of damaged cells for low-flow push-pull perfusion. Modeling revealed that shear stress and pressure gradients generated by the flow were lower than thresholds expected to cause damage. Comparison with existing methods.Push-pull perfusion caused less tissue damage but yielded 1500-fold better spatial resolution.

Conclusions: Push-pull perfusion at low flow rates is a viable method for sampling from the brain with potential for high temporal and spatial resolution. Tissue damage is mostly caused by probe insertion. Smaller probes may yield even lower damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jneumeth.2015.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331210PMC
March 2015

Hyperglycemia- and neuropathy-induced changes in mitochondria within sensory nerves.

Ann Clin Transl Neurol 2014 Oct 1;1(10):799-812. Epub 2014 Oct 1.

Division on Metabolism Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan Ann Arbor, Michigan, 48105.

Objective: This study focused on altered mitochondrial dynamics as a potential mechanism for diabetic peripheral neuropathy (DPN). We employed both an in vitro sensory neuron model and an in situ analysis of human intraepidermal nerve fibers (IENFs) from cutaneous biopsies to measure alterations in the size distribution of mitochondria as a result of hyperglycemia and diabetes, respectively.

Methods: Neurite- and nerve-specific mitochondrial signals within cultured rodent sensory neurons and human IENFs were measured by employing a three-dimensional visualization and quantification technique. Skin biopsies from distal thigh (DT) and distal leg (DL) were analyzed from three groups of patients; patients with diabetes and no DPN, patients with diabetes and confirmed DPN, and healthy controls.

Results: This analysis demonstrated an increase in mitochondria distributed within the neurites of cultured sensory neurons exposed to hyperglycemic conditions. Similar changes were observed within IENFs of the DT in DPN patients compared to controls. This change was represented by a significant shift in the size frequency distribution of mitochondria toward larger mitochondria volumes within DT nerves of DPN patients. There was a length-dependent difference in mitochondria within IENFs. Distal leg IENFs from control patients had a significant shift toward larger volumes of mitochondrial signal compared to DT IENFs.

Interpretation: The results of this study support the hypothesis that altered mitochondrial dynamics may contribute to DPN pathogenesis. Future studies will examine the potential mechanisms that are responsible for mitochondrial changes within IENFs and its effect on DPN pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/acn3.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241807PMC
October 2014

Differential reduction in corneal nerve fiber length in patients with type 1 or type 2 diabetes mellitus.

J Diabetes Complications 2014 Sep-Oct;28(5):658-61. Epub 2014 Jun 17.

Department of Ophthalmology and Visual Sciences, University of Michigan Medical School. Electronic address:

Aim: To examine the relationship between corneal nerve fiber length (CNFL) and diabetic neuropathy (DN) status in patients with type 1 or type 2 diabetes mellitus (DM).

Methods: In this cross-sectional study, we examined 25 diabetic patients without DN, 10 patients with mild DN, 8 patients with severe DN, and 9 controls without diabetes. DN status was assigned based on a combination of clinical symptoms, signs, and electrophysiological testing. Patients underwent corneal confocal microscopy (CCM) of the sub-basal nerve plexus. Post-hoc analysis of the CCM images was performed to quantify the average CNFL, and ANOVA was used to assess for differences in CNFL.

Results: All 25 subjects without DN had type 1 DM, and subjects with DN had type 2 DM. Participants with severe DN had significantly lower CNFL (12.5±6.1mm/mm(2)) compared to controls (20.7±2.2mm/mm(2)) (p=0.009). However, lower CNFL was also found in participants with type 1 DM who did not have DN (15.1±4.7mm/mm(2)) relative to controls (p=0.033).

Conclusions: CCM of the sub-basal nerve plexus may be an indicator of early peripheral nerve degeneration in type 1 DM. Type of diabetes, in addition to degree of neuropathy, may influence the extent of corneal nerve damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jdiacomp.2014.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146399PMC
June 2015

Quantifying size and number of adipocytes in adipose tissue.

Methods Enzymol 2014 ;537:93-122

Department of Molecular and Integrative Physiology, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA; Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA. Electronic address:

White adipose tissue (WAT) is a dynamic and modifiable tissue that develops late during gestation in humans and through early postnatal development in rodents. WAT is unique in that it can account for as little as 3% of total body weight in elite athletes or as much as 70% in the morbidly obese. With the development of obesity, WAT undergoes a process of tissue remodeling in which adipocytes increase in both number (hyperplasia) and size (hypertrophy). Metabolic derangements associated with obesity, including type 2 diabetes, occur when WAT growth through hyperplasia and hypertrophy cannot keep pace with the energy storage needs associated with chronic energy excess. Accordingly, hypertrophic adipocytes become overburdened with lipids, resulting in changes in the secreted hormonal milieu. Lipids that cannot be stored in the engorged adipocytes become ectopically deposited in organs such as the liver, muscle, and pancreas. WAT remodeling therefore coincides with obesity and secondary metabolic diseases. Obesity, however, is not unique in causing WAT remodeling: changes in adiposity also occur with aging, calorie restriction, cancers, and diseases such as HIV infection. In this chapter, we describe a semiautomated method of quantitatively analyzing the histomorphometry of WAT using common laboratory equipment. With this technique, the frequency distribution of adipocyte sizes across the tissue depot and the number of total adipocytes per depot can be estimated by counting as few as 100 adipocytes per animal. In doing so, the method described herein is a useful tool for accurately quantifying WAT development, growth, and remodeling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/B978-0-12-411619-1.00006-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069255PMC
September 2014

Adenylyl cyclase 6 mediates the action of cyclic AMP-dependent secretagogues in mouse pancreatic exocrine cells via protein kinase A pathway activation.

J Physiol 2013 Aug 10;591(15):3693-707. Epub 2013 Jun 10.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA.

Both secretin and vasoactive intestinal polypeptide (VIP) receptors are responsible for the activation of adenylyl cyclases (ACs), which increase intracellular cyclic AMP (cAMP) levels in the exocrine pancreas. There are nine membrane-associated isoforms, each with its own pattern of expression and regulation. In this study we sought to establish which AC isoforms play a regulatory role in pancreatic exocrine cells. Using RT-PCR, AC3, AC4, AC6, AC7 and AC9 were found to be expressed in the pancreas. AC3, AC4, AC6 and AC9 were expressed in both pancreatic acini and ducts, whereas AC7 was expressed only in pancreatic ducts. Based on known regulation by intracellular signals, selective inhibitors and stimulators were used to suggest which isoforms play an important role in the induction of cAMP formation. AC6 appeared to be an important isoform because protein kinase A (PKA), PKC and calcium all inhibited VIP-induced cAMP formation, whereas calcineurin or calmodulin did not modify the response to VIP. Mice with genetically deleted AC6 were studied and showed reduced cAMP formation and PKA activation in both isolated pancreatic acini and duct fragments. The absence of AC6 reduced cAMP-dependent secretagogue-stimulated amylase secretion, and abolished fluid secretion in both in vivo and isolated duct fragments. In conclusion, several AC isoforms are expressed in pancreatic acini and ducts. AC6 mediates a significant part of pancreatic amylase and fluid secretion in response to secretin, VIP and forskolin through cAMP/PKA pathway activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/jphysiol.2012.249698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3752451PMC
August 2013

Three-dimensional imaging of nociceptive intraepidermal nerve fibers in human skin biopsies.

J Vis Exp 2013 Apr 29(74):e50331. Epub 2013 Apr 29.

Department of Neurology, University of Michigan, MI, USA.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy (1,2). At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies (3). However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma. In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy (4). In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor (5). The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/50331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3667646PMC
April 2013

Vulnerability of the retinal microvasculature to oxidative stress: ion channel-dependent mechanisms.

Am J Physiol Cell Physiol 2012 May 15;302(9):C1413-20. Epub 2012 Feb 15.

Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan 48505, USA.

Although oxidative stress is a hallmark of important vascular disorders such as diabetic retinopathy, it remains unclear why the retinal microvasculature is particularly vulnerable to this pathophysiological condition. We postulated that redox-sensitive ion channels may play a role. Using H(2)O(2) to cause oxidative stress in microvascular complexes freshly isolated from the adult rat retina, we assessed ionic currents, cell viability, intracellular oxidants, and cell calcium by using perforated-patch recordings, trypan blue dye exclusion, and fura-2 fluorescence, respectively. Supporting a role for the oxidant-sensitive ATP-sensitive K (K(ATP)) channels, we found that these channels are activated during exposure of retinal microvessels to H(2)O(2). Furthermore, their inhibition by glibenclamide significantly lessened H(2)O(2)-induced microvascular cell death. Additional experiments established that by increasing the influx of calcium into microvascular cells, the K(ATP) channel-mediated hyperpolarization boosted the vulnerability of these cells to oxidative stress. In addition to the K(ATP) channel-dependent mechanism for increasing the lethality of oxidative stress, we also found that the vulnerability of cells in the capillaries, but not in the arterioles, was further boosted by a K(ATP) channel-independent mechanism, which our experiments indicated involves the oxidant-induced activation of calcium-permeable nonspecific cation channels. Taken together, our findings support a working model in which both K(ATP) channel-independent and K(ATP) channel-dependent mechanisms render the capillaries of the retina particularly vulnerable to oxidative stress. Identification of these previously unappreciated mechanisms for boosting the lethality of oxidants may provide new targets for pharmacologically limiting damage to the retinal microvasculature during periods of oxidative stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpcell.00426.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361947PMC
May 2012

Visualization of mitochondrial DNA replication in individual cells by EdU signal amplification.

J Vis Exp 2010 Nov 15(45). Epub 2010 Nov 15.

Michigan Research Community, Undergraduate Research Opportunity Program, University of Michigan, USA.

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/2147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159597PMC
November 2010

CCK-independent mTORC1 activation during dietary protein-induced exocrine pancreas growth.

Am J Physiol Gastrointest Liver Physiol 2010 Nov 26;299(5):G1154-63. Epub 2010 Aug 26.

Department of 1Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

Dietary protein can stimulate pancreatic growth in the absence of CCK release, but there is little data on the regulation of CCK-independent growth. To identify mechanisms whereby protein stimulates pancreatic growth in the absence of CCK release, C57BL/6 control and CCK-null male mice were fed normal-protein (14% casein) or high-protein (75% casein) chow for 7 days. The weight of the pancreas increased by 32% in C57BL/6 mice and 26% in CCK-null mice fed high-protein chow. Changes in pancreatic weight in control mice were due to both cell hypertrophy and hyperplasia since there was an increase in protein-to-DNA ratio, total DNA content, and DNA synthesis. In CCK-null mice pancreatic growth was almost entirely due to hypertrophy with both protein-to-DNA ratio and cell size increasing without significant increases in DNA content or DNA synthesis. ERK, calcineurin, and mammalian target of rapamycin complex 1 (mTORC1) are activated in models of CCK-induced growth, but there were no differences in ERK or calcineurin activation between fasted and fed CCK-null mice. In contrast, mTORC1 activation was increased after feeding and the duration of activation was prolonged in mice fed high-protein chow compared with normal-protein chow. Changes in pancreatic weight and RNA content were completely inhibited, and changes in protein content were partially abated, when the mTORC1 inhibitor rapamycin was administered during high-protein chow feeding. Prolonged mTORC1 activation is thus required for dietary protein-induced pancreatic growth in the absence of CCK.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpgi.00445.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2993171PMC
November 2010

Mitochondrial DNA (mtDNA) biogenesis: visualization and duel incorporation of BrdU and EdU into newly synthesized mtDNA in vitro.

J Histochem Cytochem 2010 Feb;58(2):207-18

Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, Michigan 48109, USA.

Mitochondria are key regulators of cellular energy and are the focus of a large number of studies examining the regulation of mitochondrial dynamics and biogenesis in healthy and diseased conditions. One approach to monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive technique to visualize newly synthesized mtDNA in individual cells to study mtDNA replication within subcellular compartments of neurons. The technique combines the incorporation of 5-bromo-2-deoxyuridine (BrdU) and/or 5-ethynyl-2'-deoxyuridine (EdU) into mtDNA, together with a tyramide signal amplification protocol. Employing this technique, we visualized and measured mtDNA biogenesis in individual cells. The labeling procedure for EdU allows for more comprehensive results by allowing the comparison of its incorporation with other intracellular markers, because it does not require the harsh acid or enzyme digests necessary to recover the BrdU epitope. In addition, the utilization of both BrdU and EdU permits sequential pulse-chase experiments to follow the intracellular localization of mtDNA replication. The ability to quantify mitochondrial biogenesis provides an essential tool for investigating the alterations in mitochondrial dynamics involved in the pathogenesis of multiple cellular disorders, including neuropathies and neurodegenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1369/jhc.2009.954701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2803709PMC
February 2010

Hydrogen peroxide-induced Akt phosphorylation regulates Bax activation.

Biochimie 2009 May 6;91(5):577-85. Epub 2009 Feb 6.

Department of Neurology, University of Michigan, Ann Arbor, MI 48109-2200, USA.

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) are involved in many cellular processes that positively and negatively regulate cell fate. H(2)O(2), acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H(2)O(2) was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H(2)O(2)-induced activation of PI3K/Akt influences post-translational modification of Bax and inactivates a key component of the cell death machinery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biochi.2009.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2697849PMC
May 2009

Criteria for creating and assessing mouse models of diabetic neuropathy.

Curr Drug Targets 2008 Jan;9(1):3-13

University of Michigan, Departments of Neurology and Internal Medicine, USA.

Diabetic neuropathy (DN) is a serious and debilitating complication of both type 1 and type 2 diabetes. Despite intense research efforts into multiple aspects of this complication, including both vascular and neuronal metabolic derangements, the only treatment remains maintenance of euglycemia. Basic research into the mechanisms responsible for DN relies on using the most appropriate animal model. The advent of genetic manipulation has moved mouse models of human disease to the forefront. The ability to insert or delete genes affected in human patients offers unique insight into disease processes; however, mice are still not humans and difficulties remain in interpreting data derived from these animals. A number of studies have investigated and described DN in mice but it is difficult to compare these studies with each other or with human DN due to experimental differences including background strain, type of diabetes, method of induction and duration of diabetes, animal age and gender. This review describes currently used DN animal models. We followed a standardized diabetes induction protocol and designed and implemented a set of phenotyping parameters to classify the development and severity of DN. By applying standard protocols, we hope to facilitate the comparison and characterization of DN across different background strains in the hope of discovering the most human like model in which to test potential therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026946PMC
http://dx.doi.org/10.2174/138945008783431763DOI Listing
January 2008

Mouse models of diabetic neuropathy.

Neurobiol Dis 2007 Dec 31;28(3):276-85. Epub 2007 Jul 31.

Department of Neurology, University of Michigan, Ann Arbor, MI 48109-2200, USA.

Diabetic neuropathy (DN) is a debilitating complication of type 1 and type 2 diabetes. Rodent models of DN do not fully replicate the pathology observed in human patients. We examined DN in streptozotocin (STZ)-induced [B6] and spontaneous type 1 diabetes [B6Ins2(Akita)] and spontaneous type 2 diabetes [B6-db/db, BKS-db/db]. Despite persistent hyperglycemia, the STZ-treated B6 and B6Ins2(Akita) mice were resistant to the development of DN. In contrast, DN developed in both type 2 diabetes models: the B6-db/db and BKS-db/db mice. The persistence of hyperglycemia and development of DN in the B6-db/db mice required an increased fat diet while the BKS-db/db mice developed severe DN and remained hyperglycemic on standard mouse chow. Our data support the hypothesis that genetic background and diet influence the development of DN and should be considered when developing new models of DN.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nbd.2007.07.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730836PMC
December 2007

Receptor-mediated regulation of tomosyn-syntaxin 1A interactions in bovine adrenal chromaffin cells.

J Biol Chem 2007 Aug 1;282(31):22887-99. Epub 2007 Jun 1.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109, USA.

Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M701787200DOI Listing
August 2007

Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo.

J Biol Chem 2004 Dec 15;279(53):55924-36. Epub 2004 Oct 15.

Department of Molecular and Integrative Physiology, The Medical School, University of Michigan, 7808 Medical Sciences II Building, Ann Arbor, MI 48109, USA.

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M410024200DOI Listing
December 2004