Rerouting mechanisms: polyol pathway and macromolecule glycation

Full article in English: Dopaminergic neurodegeneration in a rat model of long-term hyperglycemia: preferential degeneration of the nigrostriatal motor pathway

Abstract

Epidemiological evidence suggests a correlation between diabetes and age-related neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Hyperglycemia causes oxidative stress in vulnerable tissues such as the brain.
We recently demonstrated that elevated levels of glucose lead to the death of dopaminergic neurons in culture through oxidative mechanisms. Considering the lack of literature addressing dopaminergic alterations in diabetes with age, the goal of this study was to characterize the state of two critical dopaminergic pathways in the nicotinamidestreptozotocin rat model of long-term hyperglycemia, specifically the nigrostriatal motor pathway and the reward-associated mesocorticolimbic pathway. Neuronal and glial alterations were evaluated 3 and 6 months after hyperglycemia induction, demonstrating preferential degeneration of the nigrostriatal pathway complemented by a noticeable astrogliosis and loss of microglial cells throughout aging. Behavioral tests confirmed the existence of motor impairments in hyperglycemic rats that resemble early parkinsonian syrnptomatology in rats, ensuing from nigrostriatal alterations. These results solidify the relation between hyperglycemia and nigrostriatal dopaminergic neurodegeneration, providing new insight on the higher occurrence of Parkinson’s disease in diabetic patients.

Introduction

Glucose is the obligate energy substrate of adult neurons. Owing to the preponderant expression of glucose transporter 1 (GLUTl) at the blood-brain barrier (Anraku et al., 2017) and GLUT3 at the neuron plasma membrane (Patching et al., 2017, Simpson et al., 2007), uptake overwhelmingly occurs in an insulinindependent fashion. Thus, intraneuronal glucose concentrations directly depend on extracellular concentrations, i.e., on plasma glucose concentrations (Jacob et al., 2002).
Indeed, neurons belong to the most severely hit targets in hyperglycemia, along with mesangial and capillary endothelial ceUs (for review see Brownlee, 2005).
Well appreciated is the fact that persistent hyperglycemia induces oxidative stress occasioning damage in the peripheral nervous system, as is the case in comorbid neuropathies of uncorttrolled diabetes (Rajabally, 2017). Fewer studies have focused their attention on hyperglycemia in the human central nervous system (Moulton et al., 2015) but go as far as to draw relationships between diabetes and age-related neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases (Jagota et al., 2012, Vicente Miranda et al., 2016, Vieira et al., 2017, Vignini et al., 2013).
Notwithstanding the wealth of evidence supporting a toxic role for glucose ln neurons of the central nervous system (see for review Tomlinson and Gardiner 2008, Vicente Miranda et al., 2016), details are lacking in the bigger picture. Most studies have addressed neurodegeneration in discrete brain regions on a short-term scale (Do Nascimento et al., 2011, Kamboj and Sandhir, 2011), which do not represent temporal or spatial breadths of hyperglycemic distress in aging diabetic patients. Moreover, literature describing dopaminergic neurodegeneration in hyperglycemic models remains modest. Our group demonstrated a role for chronic glucose exposure in apoptotic death of dopaminergic neurons through oxidative mechanisms (Bournival et al., 2012, Renaud et al., 2014) and several studies report dopaminergic alterations in diabetes or acute hyperglycemia (Lozovsky et al., 1981 , Murzi et al., 1996, Sevak et al., 2007). Nevertheless, the state of entire dopaminergic pathways in the long-term has not been inquired.
In order to improve our understanding of central dopaminergic neurodegeneration in hyperglycemia, we focused on two critical central dopaminergic systems, namely the nigrostriatal and mesocorticolimbic pathways (for review see Haber, 2014).
The nigrostriatal pathway is primarily involved in the generation of movement. Cell bodies of nigrostriatal neurons are found in the substantia nigra pars compacta (SNe), lodged in the ventral midbrain, and extend their dopaminergic fiber terminaIs to the dorsal striatum (DS). The mesocorticolimbic pathway is often termed the reward system. Cell bodies of mesocorticolimbic neurons are also found in the ventral midbrain, though in a neighboring subregion named the ventral tegmental area (VT A).
This pathway innervates both the ventral striatum (VS, mesolimbic pathway), which contains the nucleus accumbens (NAcc) and the olfactory tubercle (ûT), as weil as the prefrontal cortex (PFC, mesocortical pathway) (Squire et al., 2008). We thus evaluated neurodegeneration and analyzed glial populations in these regions to obtain a better appreciation of the global state of the dopaminergic pathways in long-term hyperglycemia. Finally, behavioral assessments were carried out to uncover motor and cognitive alterations possibly expressed as a consequence of neurodegeneration. Research design and methods.

Subjects

A total of 91 rats were used ln this study. For immunoblotting, immunohistochemical and behavioral studies, two different cohorts totaling 61 male Sprague-Dawley rats (Charles River, St-Constant, Canada) weighing 175-200 g and aged 5-6 weeks were housed under standard laboratory conditions (12 h light/darkcycle). In Italy, motor and cognitive behavior analyses were repeated and complementary microdialysis studies were carried out in 30 male Sprague-Dawley rats (Harlan Italy, Udine, Italy) of the same weight and age, also housed under standard laboratory conditions. In all experiments, standard food and water were available ad libitum. Rats were acclimated for 2 weeks and were therefore 7 -8 weeks of age upon induction of hyperglycemia described below. AH experiments were conducted in accordance with the guidelines for animal experimentation of the EU directives (2010/63/EU; L.276; 22/09/2010), of the Ethical Committee of the University of Cagliari, of the AnimaIs for Research Act and following the legislation and policies of the Canadian Council on Animal Care, as well as with the guidelines established by the Animal Care Committee of the Université du Québec (Trois-Rivières) (2014-M.G.M.5). Maximal efforts were made to minimize discomfort and numbers of animaIs used.

Induction of long-term hyperglycemia

Rats were randomly divided Ïnto two groups; control (CTRL) and hyperglycemic (HG). Long-term hyperglycemia was induced in fasted rats (overnight, 12-16 h) by a single i.p. injection of freshly dissolved streptozotocin (STZ, 0.1 M cold citrate buffer, pH 4.5, 55 mg/kg b.w.). Nicotinamide (NA, 100 mg/kg b.w.) dissolved in physiological saline was administered i.p. exactly 20 min prior to the STZ injection to minimize the destruction of insulin-producing pancreatic beta cells, yielding HG rats that would survive for 6 months without the need for glycemia-Iowering treatments (Badole et al., 2015, Masiello et al., 1998). CTRL rats received i.p. injections ofvehicles. All rats were injected on mornings within a time frame of 3 h. Hyperglycemia was confirmed 72 h after NA-STZ injections using a digital glucose meter (UltraMini with One Touch Ultra strips) to analyze blood collected from the tail vein. Only rats with a glycemia steadily above 10 mM were used in this study. Exhaustive metabolic follow-ups were conducted regularly throughout experiments (Supplementary Figure SI).
Motor behavior assessments.We investigated the possibility that the degeneration of neurons and neuronal fibers may be accompanied by alterations in motor behavior. Rats were submitted to motor behavior assessments, beginning with baseline evaluations prior to hyperglycemia induction, followed by trials 3 and 6 months later.

Immunohistochemistry

Frozen post-fixed brain hemispheres were cut into 20 I-lm-thick coronal freefloating seriaI sections. By systematic random sampling (West, 2012), one out of every six sections was immunoreacted with antibodies raised against either the specific dopaminergic neuron marker anti-tyrosine hydroxylase (anti-TH), the general neuronal marker anti-NeuN, the astrocyte marker anti-glial fibriUary acidic protein (anti-GFAP), or the microglial ceU marker anti-ionized calcium-binding adapter molecule l (antiThal) (for list of antibodies, concentrations and manufacturers see Supplementary Table SI). Then, sections were incubated with a HRP-conjugated secondary antibody, revealed with 3,3′ -diaminobenzidine, mounted on microscope slides, dehydrated, and analyzed under a microscope in brightfield mode (MBF Bioscience, WiUiston, VT, USA). Neuroanatomical loci were determined according to the atlas of Paxinos and Watson (1998) and were identicaUy delimited in aU sections of the same anteriority between animaIs. Because of the systematic random sampling method, the same numbers of sections at the same anteriorities per region were consistently analyzed in each animal, foUowing guidelines already established for stereology (West, 2012).
Total counts of overaU neurons (NeuN+), dopaminergic neurons (TH+), astrocytes (GFAP+) and microglial ceUs (Tha1+) were performed in the SNc and in the VTA (bregma -4.80 to -6.30 mm) using the manual ceU counting marker function provided by the NIH ImageJ software version 1.49. In the DS (lateral and media l, bregma 2.2 to -1.0 mm), the VS (NAcc and OT, bregma 2.7 to 0.5 mm), the PFC (bregma 4.0 to 2.0 mm) and the HPC (bregma -2.3 to -6.0 mm), we counted total numbers of NeuN+, GF AP+ and Iba+ cells. Density of TH+ dopaminergic terminais in the striatum (Iateral, medial, NAcc and OT, refer to aforementioned bregmas) was evaluated by densitometric assays using ImageJ. These were systematically performed on non-overlapping spherical regions of interest (3 x 1 mm2 for lateral or medial DS; 5 x 0.5 mm2 for NAcc or OT).

Immunoblotting

Frozen tissues were homogenized by steel bail milling in RIPA buffer and proteins were quantified by the bicinchoninic acid method before running them in a SDS-PAGE.
Proteins were transferred cnte polyvinylidene difluoride membranes, which in tum were incubated with primary antibodies raised against the dopaminergic markers TH or dopamine transporter (DAT), or against the general neuronal marker NeuN. Following incubation with HRP-conjugated secondary antibodies, blots were finally developed with an enhanced chemiluminescence substrate solution and immunopositive chemiluminescent signaIs were visualized using the AlphaEase FC imaging system and software (San Leandro, CA, USA). Densitometric blot analyses were performed using ImageJ. ~-tubulin blots served as a loading comparative standard and ail immunoblotting results have been accordingly normalized to ~-tubulin levels.

Table des matières

ACKNOWLEDGMENTS
PREFACE
RÉSUMÉ
SUMMARY 
LIST OF T ABLES 
LIST OF FIGURES 
LIST OF ABBREVIATIONS AND ACRONYMS 
CHAPTER I
INTRODUCTION 
1.1 Dopaminergic neurons
1.1.1 Dopamine metabolism and neurotransmission
1.1.2 Central dopaminergic systems
1.1.2.1 The nigrostriatal pathway
1.1.2.2 The mesocorticolimbic pathway
1.1.2.3 Converging and diverging functions
1.1.3 Parkinson ‘s disease
1.1.3.1 Clinical symptomatology and treatments
1.1.3.2 Prodrome and biomarkers
1.1.3.3 Pathophysiology
1.1.3.4 Vulnerability of the nigrostriatal pathway
1.2 Hyperglycaemia in the central nervous system
1.2.1 Glucose as a preferential fuel for the brain
1.2.2 Neuronal glucose transport
1.2.2.1 Transporters and kinetics
1.2.2.2 Physiological considerations
1.2.3 Neuronal glucose metabolism
1.2.3.1 Specifie metabolic fates
1.2.3.2 CUITent hypotheses in neuroenergetics
l.2.4 Neuronal oxidative stress in hyperglycaemia
1.2.4.1 Mitochondrial mechanisms
1.2.4.2 Rerouting mechanisms: polyol pathway and macromolecule glycation
l.2.5 Vulnerability of the nigrostriatal pathway
1.2.5.1 Epidemiological basis: Parkinson ‘s disease in diabetic patients
1.2.5.2 Molecular and cellular bases
1.3 The antioxidative polyphenol resveratrol
1.3.1 Background
1.3.1.1 Historical context
1.3.1.2 Dietary origins
1.3.1.3 Structure, chemistry and antioxidative functions
1.3.2 Protection of dopaminergic neurons against oxidative stress
1.3.3 Direct putative targets
1.3.3.1 Ribosyldihydronicotinamide dehydrogenase (quinone) and oxidative stress
1.3.3.2 Phosphodiesterases and the energy sensing axis
1.3.3.3 Mammalian target of rapamycin and autophagy
1.3.3.4 Other targets
1.4 Research aims and hypotheses
1.4.1 Objective 1: Evaluate the degeneration of cultured dopaminergic neuronal cells in high glucose conditions
1.4.2 Objective 2: Determine the potential of the antioxidative polyphenol resveratrol to hamper the high glucose-induced degeneration of cultured dopaminergic neuronal cells
1.4.3 Objective 3: Characterize dopaminergic neurodegeneration in a rat model of long-term hyperglycaemia
1.4.4 Objective 4: Assess the behavioural alterations resulting from nigrostriatal neurodegeneration in a rat model of long-term hyperglycaemia
1.5 Methodology
l.5.1 Objective 1: Evaluate the degeneration of cultured dopaminergic neuronal cells in high glucose conditions
1.5.1.1 CeU culture
1.5.1.2 High glucose conditions
1.5.1.3 Superoxide anion quantification
1.5.1.4 Evaluation of apoptotic death
1.5.2 Objective 2: Determine the potential of the antioxidative polyphenol resveratrol to hamper the high glucose-induced degeneration of cultured dopaminergic neuronal ceUs
1.5.2.1 Resveratrol treatments
1.5.3 Objective 3: Characterize dopaminergic neurodegeneration in a rat model of long-term hyperglycaemia
1.5.3.1 Rat model of long-term hyperglycaemia
1.5.3.2 Intracerebral glucose measurements
1.5.3.3 Assessment of neurodegeneration
1.5.3.4 Assessment of glial profiles
1.5.3.5 Intracerebral dopamine measurements
1.5.4 Objective 4: Assess the behavioural alterations resulting from nigrostriatal neurodegeneration in a rat model of long-term hyperglycaemia
1.5.4.1 Assessment of motor deficits
1.5.4.2 Evaluation of social behaviour
CHAPTER II
RESVERA TROL PROTECTS DOP AMINERGIC PC12 CELLS FROM HIGH GLUCOSE-INDUCED OXIDA TIVE STRESS AND APOPTOSIS: EFFECT ON P53 AND GLUCOSE-REGULATED PROTEIN  LOCALIZA TION 
2.1 Author contributions
2.2 Résumé
2.3 Full article in English: Resveratrol protects dopaminergic PC12 ce Us from high glucose induced oxidative stress and apoptosis: effect on p53 and glucose-regulated protein 75 localization
Abstract
Introduction
Materials and methods
Drugs and chemicals
Cell culture and treatments
Detection of mitochondrial superoxide radical
Immunofluorescence and terminal deoxynucleotidyl transferase
dUTP nick end labeling as say
Specific apoptotic DNA denaturation analysis
Protein extraction
Electrophoresis and Western blotting analysis
Glucose-regulated prote in 75-p53 colocalization
Statistical analysis
Results
Resveratrol rescues high glucose-induced production of superoxide
Resveratrol reduces high glucose-induced apoptosis
Resveratrol modulates p53 and glucose-regulated protein 75 subcellular localization and colocalization
Discussion
Acknowledgments
References
CHAPTER III
DOPAMINERGIC NEURODEGENERA TION IN A RA T MODEL OF LONG-TERM HYPERGL YCEMIA: PREFERENTIAL DEGENERA TION OF THE NIGROSTRIATAL MOTOR PATHWAY 
3.1 Author contributions
3.2 Résumé
3.3 Full article in English: Dopaminergic neurodegeneration in a rat model of long-term hyperglycemia: preferential degeneration of the nigrostriatal motor pathway
Abstract
Introduction
Research design and methods
Subjects
Induction of long-term hyperglycemia
Motor behavior assessments
Cognitive behavior
Sacrifices and tissue harvest
Immunohistochemistry
Immunoblotting
Intracerebral microdialysis in freely moving rats
Brain tissue and microdialysate glucose concentrations
Statistical analyses
Results
Glucose concentrations increase in aU brain regions of interest
Long-term hyperglycemia causes preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta
Long-term hyperglycemia causes preferential degeneration of dopaminergic fiber terminaIs in the dorsal striatum
Long-term hyperglycemia does not cal}se substantial neurodegeneration in the prefrontal cortex or in the hippocampus
Long-term hyperglycemic rats display astrogliosis and loss of microglial cells in degenerated dopaminergic regions
Long-term hyperglycemic rats show altered motor behaviour
Discussion
Acknowledgments
References
Supplementary data
Metabolic follow-up and disease progression
CHAPTER IV
LONG-TERM HYPERGL YCAEMIA MODIFIES SOCIAL BEHA VIOUR AND EMISSION OF ULTRASONIC VOCALISATIONS IN RATS: A POSSIBLE EXPERIMENTAL MODEL OF AL TE RED SOCIABILITY IN DIABETES 
4.1 Author contributions
4.2 Résumé
4.3 Full article in English: Altered social behaviour in long-term hyperglycemic rats displaying dopaminergic striatal denervation: an ultrasonic vocalisation study
Abstract
Introduction
Results
Occurrences of social behaviours and ultrasonic vocalisations
Behavioural covariance profile
Behaviour-vocalisation covariance profile
Magnitude of social interactivity and ultrasonic vocalisations in
relation to the degree of striatal denervation, hypoinsulinaemia and
glucose intolerance
Discussion
Methods
Subjects
Induction ofhyperglycaemia
Oral glucose intolerance test and baseline insulinemia
Ultrasonic vocalisations and social behaviour
Immunohistochemistry
Statistical analyses
Acknowledgments References
CHAPTER V
DISCUSSION 
5.1 Objectives 1 and 2: In vitro, high glucose-induced oxidative stress leads to the death of dopaminergic neurons avertible by resveratrol treatments
5.1 .1 Drawing parallels with Brownlee’s theory
5.1.1.1 From oxidative stress to apoptosis
5.1.1.2 Paradoxical poly(adenosine diphosphate-ribose) polyrnerase inactivation
5.1.1.3 Validating Brownlee’s model
5.1.2 Resveratrol: partial antioxidative effects, but full neuroprotection
5.1.3 The relevance of glucose-regulated protein 75 in Parkinson’s di sease
5.2 Objectives 3 and 4: In vivo, long-term hyperglycaemia causes preferential nigrostriatal dopaminergique neurodegeneration and consequential behavioural alterations
5.2.1 Intracerebral glucose concentrations
5.2.2 Altered glial profiles as an indicator of oxidative stress
5.2.3 Nigrostriatal dopaminergic neuronal death: beyond the validation of our hypothesis
5.2.3.1 Subtle neurodegeneration and motor deficits
5.2.3.2 Time course of neurodegeneration
5.2.4 Hyper-aggressive and hyper-sociable manifestations
5.2.4.1 A possible relationship with nigrostriatal dopaminergic neurodegeneration
5.2.4.2 A possible relationship with phasic and tonic dopaminergic neurotransmission
5.2.5 Effects attributable to hypoinsulinaemia
5.2.5.1 Insulin in neurodegeneration
5.2.5.2 Insulin in behaviour
5.2.6 Improving the model
5.3 Therapeutic perspectives
5.3.1 Implications for diabetic patients
5.3 .2 Implications for parkinsonian patients
5.3 .3 Employing resveratrol to therapeutic ends
5.4 Concluding remarks
REFERENCES 
APPENDIXA
LA NEURO-INFLAMMA TION : DR JEKYLL OU MR HYDE?………………….. 389
APPENDIXB OLD MOLECULES, NEW INSIGHTS: THERAPEUTIC CONSIDERATIONS FOR THE USE OF POL YPHENOLS IN NEURODEGENERATIVE DISEASES
APPENDIXC
PREVENTION OF NEUROINFLAMMATION BY RESVERA TROL: FOCUS ON EXPE~ENTAL MO DELS AND MOLECULAR MECHANISMS
APPENDIXD
THE LINK BETWEEN PARKINSON’S DISEASE AND ATTENTIONDEFICIT
HYPERACTIVITY DISORDER: AN ACCOUNT OF THE
EVIDENCE

Télécharger le rapport complet