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1.
Neurobiol Aging ; 132: 220-232, 2023 Dec.
Article En | MEDLINE | ID: mdl-37864952

The application of the selective allosteric M1 muscarinic and sigma-1 receptor agonist, AF710B (aka ANAVEX3-71), has shown to attenuate Alzheimer's disease-like hallmarks in McGill-R-Thy1-APP transgenic rats when administered at advanced pathological stages. It remains unknown whether preventive treatment strategies applying this compound may be equally effective. We tested whether daily oral administration of AF710B (10 µg/kg) in 7-month-old, preplaque, McGill-R-Thy1-APP rats for 7 months, followed by a 4-week washout period, could prevent Alzheimer's disease-like pathological hallmarks. Long-term AF710B treatment prevented the cognitive impairment of McGill-R-Thy1-APP rats. The effect was accompanied by a reduction in the number of amyloid plaques in the hippocampus and the levels of Aß42 and Aß40 peptides in the cerebral cortex. AF710B treatment also reduced microglia and astrocyte recruitment toward CA1 hippocampal Aß-burdened neurons compared to vehicle-treated McGill-R-Thy1-APP rats, also altering the inflammatory cytokines profile. Lastly, AF710B treatment rescued the conversion of brain-derived neurotrophic factor precursor to its mature and biologically active form. Overall, these results suggest preventive and disease-modifying properties of the compound.


Alzheimer Disease , Cognitive Dysfunction , Receptors, sigma , Rats , Animals , Mice , Alzheimer Disease/pathology , Rats, Transgenic , Amyloid beta-Protein Precursor , Amyloid beta-Peptides , Amyloidogenic Proteins , Cognitive Dysfunction/drug therapy , Cognitive Dysfunction/prevention & control , Cognitive Dysfunction/complications , Plaque, Amyloid/pathology , Disease Models, Animal , Mice, Transgenic , Sigma-1 Receptor
2.
J Neurochem ; 163(2): 149-167, 2022 10.
Article En | MEDLINE | ID: mdl-35921478

Basal forebrain cholinergic neurons (BFCNs) represent the main source of cholinergic innervation to the cortex and hippocampus and degenerate early in Alzheimer's disease (AD) progression. Phenotypic maintenance of BFCNs depends on levels of mature nerve growth factor (mNGF) and mature brain-derived neurotrophic factor (mBDNF), produced by target neurons and retrogradely transported to the cell body. Whether a reciprocal interaction where BFCN inputs impact neurotrophin availability and affect cortical neuronal markers remains unknown. To address our hypothesis, we immunolesioned the nucleus basalis (nb), a basal forebrain cholinergic nuclei projecting mainly to the cortex, by bilateral stereotaxic injection of 192-IgG-Saporin (the cytotoxin Saporin binds p75ntr receptors expressed exclusively by BFCNs) in 2.5-month-old Wistar rats. At 6 months post-lesion, Saporin-injected rats (SAP) showed an impairment in a modified version of the 5-Choice Serial Reaction Time Task (5-choice task). Postmortem analyses of the brain revealed a reduction of Choline Acetyltransferase-immunoreactive neurons compared to wild-type controls. A diminished number of cortical vesicular acetylcholine transporter-immunoreactive boutons was accompanied by a reduction in BDNF mRNA, mBDNF protein levels, markers of glutamatergic (vGluT1), and GABAergic (GAD65) neurons in the SAP-group compared to the controls. NGF mRNA, NGF precursor, and mNGF protein levels were not affected. Additionally, cholinergic markers correlated with the attentional deficit and BDNF levels. Our findings demonstrate that while cholinergic nb loss impairs cognition and reduces cortical neuron markers, it produces differential effects on neurotrophin availability, affecting BDNF but not NGF levels.


Basal Forebrain , Choline O-Acetyltransferase , Animals , Rats , Basal Forebrain/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Choline O-Acetyltransferase/metabolism , Cholinergic Agents/pharmacology , Cholinergic Neurons/metabolism , Cytotoxins , Immunoglobulin G , Rats, Wistar , RNA, Messenger/analysis , Saporins/metabolism , Vesicular Acetylcholine Transport Proteins/metabolism , Nerve Growth Factor/biosynthesis
3.
Neuropathol Appl Neurobiol ; 48(6): e12835, 2022 10.
Article En | MEDLINE | ID: mdl-35822518

AIMS: The locus coeruleus (LC) is the main source of noradrenaline (NA) in the mammalian brain and has been found to degenerate during the initial stages of Alzheimer's disease (AD). Recent studies indicate that at late stages of the amyloid pathology, LC-pathological alterations accelerate AD-like pathology progression by interfering with the neuromodulatory and anti-inflammatory properties of NA. However, the impact of LC degeneration at the earliest stages of amyloidosis on the AD-like pathology is not well understood. METHODS: The LC was lesioned in wild-type and McGill-R-Thy1-APP transgenic (APP tg) rats by administering N-(2-chloroethyl)-N-ethyl-bromo-benzylamine before amyloid plaque deposition. Cognitive deficits and AD-like neuropathological changes were measured after the LC lesion. RESULTS: Four months post-treatment, rats displayed a decrease in brain noradrenergic innervation. The LC lesion in APP tg-treated rats enhanced cognitive deficits and decreased hippocampal cholinergic innervation and neurotrophin expression. In addition, the APP tg-treated rats displayed an increased microglial and astroglial cell number in close vicinity to hippocampal amyloid-beta burdened neurons. The recruited microglia showed cellular alterations indicative of an intermediate activation state. CONCLUSIONS: Our results indicate that early LC demise aggravates the early neuroinflammatory process, cognitive impairments, cholinergic deficits and neurotrophin deregulation at the earliest stages of the human-like brain amyloidosis.


Alzheimer Disease , Amyloidosis , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/metabolism , Amyloidosis/metabolism , Amyloidosis/pathology , Animals , Cholinergic Agents/metabolism , Cognition , Disease Models, Animal , Locus Coeruleus/metabolism , Nerve Growth Factors/metabolism , Norepinephrine/metabolism , Plaque, Amyloid/pathology , Rats , Rats, Transgenic
4.
J Alzheimers Dis ; 73(2): 723-739, 2020.
Article En | MEDLINE | ID: mdl-31868669

Epidemiological, preclinical, and clinical studies have suggested a role for microdose lithium in reducing Alzheimer's disease (AD) risk by modulating key mechanisms associated with AD pathology. The novel microdose lithium formulation, NP03, has disease-modifying effects in the McGill-R-Thy1-APP transgenic rat model of AD-like amyloidosis at pre-plaque stages, before frank amyloid-ß (Aß) plaque deposition, during which Aß is primarily intraneuronal. Here, we are interested in determining whether the positive effects of microdose lithium extend into early Aß post-plaque stages. We administered NP03 (40µg Li/kg; 1 ml/kg body weight) to McGill-R-Thy1-APP transgenic rats for 12 weeks spanning the transition phase from plaque-free to plaque-bearing. The effect of NP03 on remote working memory was assessed using the novel object recognition task. Levels of human Aß38, Aß40, and Aß42 as well as levels of pro-inflammatory mediators were measured in brain-extracts and plasma using electrochemiluminescent assays. Mature Aß plaques were visualized with a thioflavin-S staining. Vesicular acetylcholine transporter (VAChT) bouton density and levels of chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin-6 (IL-6), and 4-hydroxynonenal (4-HNE) were probed using quantitative immunohistochemistry. During the early Aß post-plaque stage, we find that NP03 rescues functional deficits in object recognition, reduces loss of cholinergic boutons in the hippocampus, reduces levels of soluble and insoluble cortical Aß42 and reduces hippocampal Aß plaque number. In addition, NP03 reduces markers of neuroinflammation and cellular oxidative stress. Together these results indicate that microdose lithium NP03 is effective at later stages of amyloid pathology, after appearance of Aß plaques.


Alzheimer Disease/pathology , Alzheimer Disease/prevention & control , Citrates/therapeutic use , Lithium Compounds/therapeutic use , Neuroprotective Agents/therapeutic use , Plaque, Amyloid/pathology , Plaque, Amyloid/prevention & control , Aldehydes/metabolism , Alzheimer Disease/psychology , Amyloid beta-Peptides/metabolism , Animals , Chemokines/metabolism , Drug Compounding , Encephalitis/metabolism , Encephalitis/pathology , Hippocampus/metabolism , Hippocampus/pathology , Humans , Interleukin-6/metabolism , Memory, Short-Term/drug effects , Presynaptic Terminals/pathology , Rats , Rats, Transgenic , Recognition, Psychology , Vesicular Acetylcholine Transport Proteins/metabolism
5.
Front Pharmacol ; 10: 189, 2019.
Article En | MEDLINE | ID: mdl-30886583

This Mini Review discusses the merits and shortfalls of transgenic (tg) rodents modeling aspects of the human Alzheimer's disease (AD) pathology and their application to evaluate experimental therapeutics. It addresses some of the differences between mouse and rat tg models for these investigations. It relates, in a condensed fashion, the experience of our research laboratory with the application of anti-inflammatory compounds and S-adenosylmethionine (SAM) at the earliest stages of AD-like amyloid pathology in tg mice. The application of SAM was intended to revert the global brain DNA hypomethylation unleashed by the intraneuronal accumulation of amyloid-ß-immunoreactive material, an intervention that restored levels of DNA methylation including of the bace1 gene. This review also summarizes experimental pharmacology observations made in the McGill tg rat model of AD-like pathology by applying "nano-lithium" or a drug with allosteric M1 muscarinic and sigma 1 receptor agonistic properties (AF710B). Extremely low doses of lithium (up to 400 times lower than used in the clinic) had remarkable beneficial effects on lowering pathology and improving cognitive functions in tg rats. Likewise, AF710B treatment, even at advanced stages of the pathology, displayed remarkable beneficial effects. This drug, in experimental conditions, demonstrated possible "disease-modifying" properties as pathology was frankly diminished and cognition improved after a month of "wash-out" period. The Mini-Review ends with a discussion on the predictive value of similar experimental pharmacological interventions in current rodent tg models. It comments on the validity of some of these approaches for early interventions at preclinical stages of AD, interventions which may be envisioned once definitive diagnosis of AD before clinical presentation is made possible.

6.
Neurobiol Dis ; 127: 323-338, 2019 07.
Article En | MEDLINE | ID: mdl-30905766

The assembly of tau protein into abnormal filaments and brain cell degeneration are characteristic of a number of human neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Several murine models have been generated to better understand the mechanisms contributing to tau assembly and neurodegeneration. Taking advantage of the more elaborate central nervous system and higher cognitive abilities of the rat, we generated a model expressing the longest human tau isoform (2N4R) with the P301S mutation. This transgenic rat line, R962-hTau, exhibits the main features of human tauopathies, such as: age-dependent increase in inclusions comprised of aggregated-tau, neuronal loss, global neurodegeneration as reflected by brain atrophy and ventricular dilation, alterations in astrocytic and microglial morphology, and myelin loss. In addition, substantial deficits across multiple memory and learning paradigms, including novel object recognition, fear conditioning and Morris water maze tasks, were observed at the time of advanced tauopathy. These results support the concept that progressive tauopathy correlates with brain atrophy and cognitive impairment.


Brain/pathology , Cognitive Dysfunction/metabolism , Tauopathies/metabolism , tau Proteins/metabolism , Animals , Brain/metabolism , Cognitive Dysfunction/genetics , Cognitive Dysfunction/pathology , Disease Models, Animal , Inclusion Bodies/metabolism , Inclusion Bodies/pathology , Rats , Rats, Transgenic , Tauopathies/genetics , Tauopathies/pathology , tau Proteins/genetics
7.
Front Neurosci ; 13: 62, 2019.
Article En | MEDLINE | ID: mdl-30809111

Emerging research has re-emphasized the role of the cortical cholinergic system in the symptomology and progression of Alzheimer's disease (AD). Basal forebrain (BF) cholinergic nuclei depend on target-derived NGF for survival during development and for the maintenance of a classical cholinergic phenotype during adulthood. In AD, BF cholinergic neurons lose their cholinergic phenotype and function, suggesting an impairment in NGF-mediated trophic support. We propose that alterations to the enzymatic pathway that controls the maturation of proNGF to mature NGF and the latter's ulterior degradation underlie this pathological process. Indeed, the NGF metabolic pathway has been demonstrated to be impaired in AD and other amyloid pathologies, and pharmacological manipulation of NGF metabolism has consequences in vivo for both levels of proNGF/NGF and the phenotype of BF cholinergic neurons. The NGF pathway may also have potential as a biomarker of cognitive decline in AD, as its changes can predict future cognitive decline in patients with Down syndrome as they develop preclinical Alzheimer's pathology. New evidence suggests that the cholinergic system, and by extension NGF, may have a greater role in the progression of AD than previously realized, as changes to the BF precede and predict changes to the entorhinal cortex, as anticholinergic drugs increase odds of developing AD, and as the use of donepezil can reduce rates of hippocampal and cortical thinning. These findings suggest that new, more sophisticated cholinergic therapies should be capable of preserving the basal forebrain thus having profound positive effects as treatments for AD.

8.
Curr Alzheimer Res ; 15(13): 1220-1230, 2018.
Article En | MEDLINE | ID: mdl-30182855

BACKGROUND: Microdose lithium is protective against Alzheimer's disease (AD), although the precise mechanisms through which its protective effects are conferred remain unclear. OBJECTIVE: To further examine the effects during the earliest stages of Aß pathology, we evaluated whether NP03, a microdose lithium formulation, modulates Aß-mediated oxidative damage and neuroinflammation when applied to a rat transgenic model of AD-like amyloidosis overexpressing amyloid precursor protein (APP). METHOD: McGill-R-Thy1-APP transgenic rats and wild-type littermates were treated with NP03 or vehicle formulation for 8 weeks beginning at 3 months of age - a phase preceding Aß plaque deposition in the transgenic rats. RESULTS: Oxidative and nitrosative stress markers, protein-bound 4-hydroxynonenal (HNE) and proteinresident 3-nitrotyrosine (3-NT), inflammatory cytokines production, as well as microglial recruitment towards Aß-burdened neurons were assayed. NP03 significantly decreased cerebral HNE and 3-NT, and reduced production of pro-inflammatory cytokines in McGill-R-Thy1-APP transgenic rats. NP03 further reduced expression of microglia surface receptor Trem2 and led to a corresponding reduction in microglia recruitment towards Aß-burdened neurons in the CA1 region of the hippocampus. CONCLUSION: These results suggest that NP03 may function to slow the AD-like pathology in part by modifying oxidative/nitrosative damage and neuroinflammation, raising the possibility that low doses of microencapsulated lithium might be of therapeutic-preventive value during very early or preclinical AD.


Alzheimer Disease/complications , Alzheimer Disease/pathology , Encephalitis/drug therapy , Encephalitis/etiology , Lithium/therapeutic use , Plaque, Amyloid/metabolism , Aldehydes/metabolism , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Amyloidosis/etiology , Amyloidosis/prevention & control , Animals , CA1 Region, Hippocampal/drug effects , CA1 Region, Hippocampal/metabolism , CA1 Region, Hippocampal/pathology , Cytokines/metabolism , Disease Models, Animal , Humans , Mice, Transgenic , Mutation/genetics , Rats , Tyrosine/analogs & derivatives , Tyrosine/metabolism
9.
Alzheimers Dement ; 14(6): 811-823, 2018 06.
Article En | MEDLINE | ID: mdl-29291374

INTRODUCTION: AF710B (aka ANAVEX 3-71) is a novel selective allosteric M1 muscarinic and sigma-1 receptor agonist. In 3×Tg-AD mice, AF710B attenuates cognitive deficits and decreases Alzheimer-like hallmarks. We now report on the long-lasting disease-modifying properties of AF710B in McGill-R-Thy1-APP transgenic (Tg) rats. METHODS: Chronic treatment with AF710B (10 µg/kg) was initiated in postplaque 13-month-old Tg rats. Drug or vehicle was administered orally daily for 4.5 months and interrupted 5 weeks before behavioral testing. RESULTS: AF710B long-term treatment reverted the cognitive deficits associated with advanced Alzheimer-like amyloid neuropathology in Tg rats. These effects were accompanied by reductions in amyloid pathology and markers of neuroinflammation and increases in amyloid cerebrospinal fluid clearance and levels of a synaptic marker. Importantly, these effects were maintained following a 5-week interruption of the treatment. DISCUSSION: With M1/sigma-1 activity and long-lasting disease-modifying properties at low dose, AF710B is a promising novel therapeutic agent for treating Alzheimer's disease.


Alzheimer Disease/drug therapy , Receptors, sigma/drug effects , Spiro Compounds/pharmacology , Thiazolidines/pharmacology , Administration, Oral , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/metabolism , Animals , Brain/pathology , Cognition Disorders/pathology , Disease Models, Animal , Rats , Rats, Transgenic , Spiro Compounds/administration & dosage , Thiazolidines/administration & dosage , Sigma-1 Receptor
10.
Mol Neurodegener ; 11(1): 61, 2016 08 22.
Article En | MEDLINE | ID: mdl-27549140

BACKGROUND: Alpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson's disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples. RESULTS: Using our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples. CONCLUSIONS: These results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.


Brain/metabolism , Parkinson Disease/metabolism , alpha-Synuclein/metabolism , Animals , Biological Assay/methods , Disease Progression , HEK293 Cells , Humans , Lewy Bodies/metabolism , Mice , Parkinson Disease/diagnosis , Phosphorylation , Phosphoserine/metabolism , Protein Processing, Post-Translational/physiology
11.
Brain ; 137(Pt 9): 2493-508, 2014 Sep.
Article En | MEDLINE | ID: mdl-25062696

The neuropathological substrate of dementia in patients with Parkinson's disease is still under debate, particularly in patients with insufficient alternate neuropathology for other degenerative dementias. In patients with pure Lewy body Parkinson's disease, previous post-mortem studies have shown that dopaminergic and cholinergic regulatory projection systems degenerate, but the exact pathways that may explain the development of dementia in patients with Parkinson's disease remain unclear. Studies in rodents suggest that both the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways may functionally interact to regulate certain aspects of cognition, however, whether such an interaction occurs in humans is still poorly understood. In this study, we performed stereological analyses of the A9 and A10 dopaminergic neurons and Ch1, Ch2 and Ch4 cholinergic neurons located in the basal forebrain, along with an assessment of α-synuclein pathology in these regions and in the hippocampus of six demented and five non-demented patients with Parkinson's disease and five age-matched control individuals with no signs of neurological disease. Moreover, we measured choline acetyltransferase activity in the hippocampus and frontal cortex of eight demented and eight non-demented patients with Parkinson's disease, as well as in the same areas of eight age-matched controls. All patients with Parkinson's disease exhibited a similar 80-85% loss of pigmented A9 dopaminergic neurons, whereas patients with Parkinson's disease dementia presented an additional loss in the lateral part of A10 dopaminergic neurons as well as Ch4 nucleus basalis neurons. In contrast, medial A10 dopaminergic neurons and Ch1 and Ch2 cholinergic septal neurons were largely spared. Despite variable Ch4 cell loss, cortical but not hippocampal cholinergic activity was consistently reduced in all patients with Parkinson's disease, suggesting significant dysfunction in cortical cholinergic pathways before frank neuronal degeneration. Patients with Parkinson's disease dementia were differentiated by a significant reduction in hippocampal cholinergic activity, by a significant loss of non-pigmented lateral A10 dopaminergic neurons and Ch4 cholinergic neurons (30 and 55% cell loss, respectively, compared with neuronal preservation in control subjects), and by an increase in the severity of α-synuclein pathology in the basal forebrain and hippocampus. Overall, these results point to increasing α-synuclein deposition and hippocampal dysfunction in a setting of more widespread degeneration of cortical dopaminergic and cholinergic pathways as contributing to the dementia occurring in patients with pure Parkinson's disease. Furthermore, our findings support the concept that α-synuclein deposition is associated with significant neuronal dysfunction in the absence of frank neuronal loss in Parkinson's disease.


Cholinergic Neurons/pathology , Hippocampus/pathology , Lewy Body Disease/diagnosis , Parkinson Disease/diagnosis , Aged , Aged, 80 and over , Choline O-Acetyltransferase/metabolism , Cholinergic Neurons/enzymology , Female , Hippocampus/enzymology , Humans , Lewy Body Disease/enzymology , Lewy Body Disease/psychology , Male , Middle Aged , Parkinson Disease/enzymology , Parkinson Disease/psychology
12.
PLoS One ; 8(5): e64844, 2013.
Article En | MEDLINE | ID: mdl-23705016

Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.


Cognition Disorders/metabolism , Cognition Disorders/physiopathology , Dependovirus/genetics , Genetic Vectors/genetics , Septum of Brain/metabolism , Ventral Tegmental Area/metabolism , alpha-Synuclein/genetics , Animals , Choline O-Acetyltransferase/metabolism , Cognition Disorders/pathology , Diagonal Band of Broca/drug effects , Diagonal Band of Broca/metabolism , Diagonal Band of Broca/pathology , Diagonal Band of Broca/physiopathology , Dopamine/pharmacology , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Extracellular Space/drug effects , Extracellular Space/metabolism , Female , Gene Expression , Green Fluorescent Proteins/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/pathology , Hippocampus/physiopathology , Humans , Memory, Short-Term/drug effects , Mice, Transgenic , Microdialysis , Motor Activity/drug effects , Rats , Rats, Sprague-Dawley , Recombination, Genetic/genetics , Septum of Brain/pathology , Septum of Brain/physiopathology , Transgenes , Tyrosine 3-Monooxygenase/metabolism , Ventral Tegmental Area/drug effects , Ventral Tegmental Area/pathology , Ventral Tegmental Area/physiopathology
13.
J Neurochem ; 124(3): 336-46, 2013 Feb.
Article En | MEDLINE | ID: mdl-23004566

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post-mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used (15) N-choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before (1) H-NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT-mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real-time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non-invasive manner using NMR spectroscopic techniques.


Acetylcholine/biosynthesis , Choline O-Acetyltransferase/physiology , Cholinergic Neurons/metabolism , Hippocampus/chemistry , Magnetic Resonance Spectroscopy/methods , Acetylcholine/chemistry , Animals , Choline O-Acetyltransferase/chemistry , Cholinergic Neurons/enzymology , Female , Hippocampus/cytology , Humans , Magnetic Resonance Spectroscopy/standards , Nitrogen Isotopes , Protons , Radioligand Assay/methods , Radioligand Assay/standards , Rats , Rats, Sprague-Dawley , Reference Standards , Reproducibility of Results , Translational Research, Biomedical/methods
14.
Curr Top Behav Neurosci ; 11: 169-98, 2012.
Article En | MEDLINE | ID: mdl-22076698

Magnetic resonance spectroscopy (MRS) is a non-invasive technique that can be used to detect and quantify multiple metabolites. This chapter will review some of the applications of MRS to the study of brain functions. Typically, (1)H-MRS can detect metabolites reflecting neuronal density and integrity, markers of energy metabolism or inflammation, as well as neurotransmitters. The complexity of the proton spectrum has however led to the development of other nuclei-based methods, such as (31)P- and (13)C-MRS, which offer a broader chemical shift range and therefore can provide more detailed information at the level of single metabolites. The versatility of MRS allows for a wide range of clinical applications, of which neurodegeneration is an interesting target for spectroscopy-based studies. In particular, MRS can identify patterns of altered brain chemistry in Alzheimer's patients and can help establish differential diagnosis in Alzheimer's and Parkinson's diseases. Using MRS to follow less abundant neurotransmitters is currently out of reach and will most likely depend on the development of methods such as hyperpolarization that can increase the sensitivity of detection. In particular, dynamic nuclear polarization has opened up a new and exciting area of medical research, with developments that could greatly impact on the real-time monitoring of in vivo metabolic processes in the brain.


Alzheimer Disease/pathology , Aspartic Acid/analogs & derivatives , Brain/metabolism , Magnetic Resonance Spectroscopy , Parkinson Disease/pathology , Aspartic Acid/metabolism , Humans , Isotopes , Protons
15.
J Am Chem Soc ; 131(44): 16014-5, 2009 Nov 11.
Article En | MEDLINE | ID: mdl-19848401

Chemical shifts of protons can report on metabolic transformations such as the conversion of choline to phosphocholine. To follow such processes in vivo, magnetization can be enhanced by dynamic nuclear polarization (DNP). We have hyperpolarized in this manner nitrogen-15 spins in (15)N-labeled choline up to 3.3% by irradiating the 94 GHz electron spin resonance of admixed TEMPO nitroxide radicals in a magnetic field of 3.35 T during ca. 3 h at 1.2 K. The sample was subsequently transferred to a high-resolution magnet, and the enhanced polarization was converted from (15)N to methyl- and methylene protons, using the small (2,3)J((1)H,(15)N) couplings in choline. The room-temperature lifetime of nitrogen polarization in choline, T(1)((15)N) approximately 200 s, could be considerably increased by partial deuteration of the molecule. This procedure enables studies of choline metabolites in vitro and in vivo using DNP-enhanced proton NMR.


Choline/metabolism , Magnetic Resonance Spectroscopy/methods , Isotope Labeling , Kinetics , Magnetics , Nitrogen Isotopes , Protons , Spin Labels
16.
J Neurochem ; 111(2): 355-67, 2009 Oct.
Article En | MEDLINE | ID: mdl-19686242

Viral vector-mediated gene transfer is emerging as a novel therapeutic approach with clinical utility in treatment of Parkinson's disease. Recombinant adeno-associated viral (rAAV) vector in particular has been utilized for continuous l-3,4 dihydroxyphenylalanine (DOPA) delivery by expressing the tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) genes which are necessary and sufficient for efficient synthesis of DOPA from dietary tyrosine. The present study was designed to determine the optimal stoichiometric relationship between TH and GCH1 genes for ectopic DOPA production and the cellular machinery involved in its synthesis, storage, and metabolism. For this purpose, we injected a fixed amount of rAAV5-TH vector and increasing amounts of rAAV5-GCH1 into the striatum of rats with complete unilateral dopamine lesion. After 7 weeks the animals were killed for either biochemical or histological analysis. We show that increasing the availability of 5,6,7,8-tetrahydro-l-biopterin (BH4) in the same cellular compartment as the TH enzyme resulted in better efficiency in DOPA synthesis, most likely by hindering inactivation of the enzyme and increasing its stability. Importantly, the BH4 synthesis from ectopic GCH1 expression was saturable, yielding optimal TH enzyme functionality between GCH1 : TH ratios of 1 : 3 and 1 : 7.


Dopamine/biosynthesis , GTP Cyclohydrolase/genetics , Genetic Therapy/methods , Parkinsonian Disorders/therapy , Tyrosine 3-Monooxygenase/genetics , Animals , Biopterins/analogs & derivatives , Biopterins/metabolism , Corpus Striatum/metabolism , Corpus Striatum/pathology , Dependovirus/genetics , Dihydroxyphenylalanine/metabolism , Dopamine/metabolism , Female , GTP Cyclohydrolase/metabolism , Immunohistochemistry , Oxidopamine , Parkinsonian Disorders/chemically induced , Parkinsonian Disorders/metabolism , Parkinsonian Disorders/pathology , Rats , Rats, Sprague-Dawley , Sympatholytics , Tyrosine 3-Monooxygenase/metabolism
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