Recent Advances in Cholinergic Imaging and Cognitive Decline—Revisiting the Cholinergic Hypothesis of Dementia

Purpose of Review Although the cholinergic hypothesis of dementia provided a successful paradigm for the development of new drugs for dementia, this hypothesis has waned in popularity. Cholinergic brain imaging may provide novel insights into the viability of this hypothesis. Recent Findings Cholinergic receptor and forebrain volumetric studies suggest an important role of the cholinergic system in maintaining brain network integrity that may deteriorate with cognitive decline in Alzheimer disease (AD) and Lewy body disorders (LBD). Bidirectional changes in regional receptor expression may suggest the presence of compensatory responses to neurodegenerative injury. Cholinergic system changes are more complex in LBD because of additional subcortical degenerations compared to AD. Cholinergic-dopaminergic interactions affect attentional, verbal learning, and executive functions, and impairments in these two transmitter systems may jointly increase the risk of dementia in Parkinson’s disease. Summary The cholinergic hypothesis is evolving from a primary focus on memory toward expanded cognitive functions modulated by regionally more complex and interactive brain networks. Cholinergic network adaptation may serve as a novel research target in neurodegeneration

New Developments in Cholinergic Imaging in Alzheimer and Lewy Body Disorders

© 2020, This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply. Purpose of Review: This paper aims to review novel trends in cholinergic neuroimaging in Alzheimer and Lewy body parkinsonian disorders. Recent Findings: The spectrum of cholinergic imaging is expanding with the availability of spatially more precise radioligands that allow assessment of previously less recognized subcortical and cortical structures with more dense cholinergic innervation. In addition, advances in MRI techniques now allow quantitative structural or functional assessment of both the cholinergic forebrain and the pedunculopontine nucleus, which may serve as non-invasive prognostic predictors. Multimodal imaging approaches, such as PET-MRI or multiligand PET, offer new insights into the dynamic and interactive roles of the cholinergic system at both local and larger-scale neural network levels. Summary: Our understanding of the heterogeneous roles of the cholinergic system in age-related diseases is evolving. Multimodal imaging approaches that provide complimentary views of the cholinergic system will be necessary to shed light on the impact of cholinergic degeneration on regional and large-scale neural networks that underpin clinical symptom manifestation in neurodegeneration

Clinicopathological investigations of the cholinergic basal forebrain in Lewy body disorders and ageing

Cholinergic dysfunction has long been associated with cognitive impairment in Alzheimer’s disease (AD). However, neuropathological and functional imaging studies have also found significant cortical cholinergic deficit in Lewy body disorders (LBD), but in a different pattern from that in AD. There is topographical cholinerigic innervation to the cortex and the hippocampus from the basal forebrain. In light of differences in cognitive deficits seen in LBD and AD, I hypothesised that cholinergic basal forebrain subregions are differentially affected in these disorders. In this thesis, novel tissue techniques have been developed for the visualisation of pathology in human post-mortem brain tissue in three-dimensions. Based on a thorough review of the literature and my personal observations, I have established a simplified subdivisional scheme of the nucleus basalis of Meynert (nbM) in the human brain. Using this scheme, a quantification of nbM cholinergic neurons and assessment of neuropathological burden were performed in a large cohort of LBD and AD cases. Severe neuronal depletion across the entire nbM was observed in LBD with cognitive impairment and relative sparing of the anterior nbM was found in AD, supporting findings from previous neuropathological and imaging studies. Further investigation was carried out in the more rostral, hippocampal-projecting cholinergic group in the vertical limb of the nucleus of the diagonal band of Broca. Significant neurodegeneration in this area was identified in LBD with cognitive impairment, but not AD, suggesting its possible role in retrieval memory function via projection to the hippocampal CA2 subfield. In the final section, it was demonstrated that lactacystin injection into the rat nbM can replicate certain pathological and clinical features of LBD with dementia and this may be a useful model for the disease. Results from these studies support my initial hypothesis regarding differential susceptibility of the basal forebrain subregions in LBD and AD.Open Acces

Imaging cholinergic function in vivo in the brain with radioiodinated stereoisomers of quinuclidinyl benzilate

This thesis evaluates the ability of (R,S)- and (R,R)-[125I]-QNB, two radioiodinated diastereoisomers of the high affinity muscarinic antagonist quinuclidinyl benzilate (QNB), to image dynamic changes in cholinergic function in the central nervous system using in vivo autoradiography. The regional uptake and retention of (R,S)-[125I]-QNB in the rat brain between 2 and 24 hours after intravenous administration was investigated to assess the utility of this technique to image muscarinic receptors in the central nervous system. Similarly, the uptake and retention of (R,R)-[125I]-QNB was investigated between 30 mins and 6 hours after administration using in vivo autoradiography and was compared to that of (R,S)-[125I]-QNB. Secondly, the sensitivity of (R,S)- and (R,R)-[125I]-QNB to dynamic changes in cholinergic neurotransmission in vivo, was assessed in conscious rats. The uptake and retention of (R,S)- and (R,R)-[125I]-QNB following a cholinergic challenge produced by administration of the long lasting AChE inhibitor heptylphysostigmine, was investigated. Regional brain levels of (R,S)- and (R,R)-[125I]-QNB in heptylphysostigmine treated animals were compared to levels in saline treated animals by in vivo autoradiography. The ability of ACh to displace (R,S)- and (R,R)-[125I]-QNB binding from rat brain sections in vitro in the presence of heptylphysostigmine was also investigated. Finally, the effects of heptylphysostigmine administration on regional cerebral blood flow were investigated using [14C]-IAP autoradiography in conscious rats. Three hypotheses were considered to account for the lack of radioligand displacement observed following heptylphysostigmine administration: 1) Heptylphysostigmine was ineffective in inhibiting AChE and raising synaptic ACh levels at the dosage used in this thesis. 2) A small amount of displacement occurred but was masked by the effects of increased cerebral blood flow. 3) (R,S)- and (R,R)-[125I]-QNB are of too high affinity for mAChRs to be displaced by endogenous neurotransmitter. The third hypothesis is viewed as the most plausible. In conclusion, (R,S)- and (R,R)-[125I]-QNB are unsuitable ligands for the detection of cholinergic function in vivo

Nicotinic Acetylcholine Receptor Signaling in Neuroprotection

Alzheimer’s disease; Neurodegenerative diseases; Nicotine; Transporter; Gli

Nicotinic Acetylcholine Receptor Signaling in Neuroprotection

Alzheimer’s disease; Neurodegenerative diseases; Nicotine; Transporter; Gli

Iodoreboxetine: the development of a novel SPECT brain imaging tracer for the noradrenaline transporter

The noradrenaline system is extensively innervated throughout the brain, implicated in the aetiology of a wide range of psychiatric conditions, and the pharmacological modulation of the noradrenaline system has had a positive influence upon the alleviation of symptoms for those suffering psychiatric or neurological disease. Clinical imaging of the brain with technologies such as Single Photon Emission Computed Tomography (SPECT) can provide clinicians and researchers with valuable information for elucidating disease aetiology, monitoring patient condition, and also confirming the mechanism of action for drug development through occupancy studies. However, to date, there is no SPECT brain imaging tracer for the noradrenaline transporter in regular clinical use. This thesis is a body of work to develop a novel SPECT brain imaging tracer for the noradrenaline transporter with compounds that are iodinated forms of reboxetine, the selective noradrenergic reuptake inhibitor and clinically used antidepressant. From the compounds synthesised and tested, the one which demonstrated the best pharmacological affinity and selectivity for the noradrenaline transporter was NKJ-64. This same compound also displayed many of the HPLC-derived properties predicting in vivo molecular behaviour that are associated with successful brain imaging tracers. Therefore, NKJ-64 was recommended for radiolabelling and further study. PCP MODEL OF THE METABOLIC HYPOFRONTALITY OBSERVED IN SCHIZOPHRENIA: The metabolic hypofunction observed in the prefrontal cortex of schizophrenic patients is mimicked in a rat model via repeated, low-dose phencyclidine administration. The neural mechanisms underlying this hypofrontality are unclear. Whilst dopaminergic transmission is classically associated with aberrant activity in schizophrenia, modulation of noradrenergic neurotransmission is correlated with the alleviation of negative symptoms. Furthermore, the noradrenaline transporter rather than the dopamine transporter functions as the dominant dopaminergic reuptake mechanism in the prefrontal cortex. It was hypothesised that repeated phencyclidine administration in the rat would induce a down-regulation of the NAT. Ligands labelled with [3H] were used for autoradiographic imaging of the noradrenaline, dopamine, and serotonin transporters in phencyclidine-treated and control groups, and [35S]-labelled oligonucleotide probes specific for the mRNA of each transporter were used for in situ hybridisation. The binding densities of the noradrenaline and dopamine transporters were unaltered in the model, however significant selective reductions of serotonin transporter binding sites were measured. The densities of mRNA for all three monoamine transporters were unaltered, so the unaltered densities of noradrenaline and dopamine transporter binding sites and the changes in serotonin transporter binding densities were not the result of altered gene expression. Although the noradrenaline transporter was not directly affected in this model of one particular aspect of schizophrenia and the model is not ideal for demonstrating the capability of a SPECT tracer for the NAT, this does not imply that the noradrenaline transporter is uninvolved in the disease aetiology of schizophrenia or of diminished importance to future schizophrenia studies. The aforementioned correlation of noradrenaline transporter modulation to the alleviation of negative symptoms emphasises the importance of acquiring the clinical ability to assess the density and/or occupancy of the noradrenaline transporter by developing a useful SPECT brain imaging tracer for this site. PHARMACOLOGICAL CHARACTERISATION OF POTENTIAL SPECT BRAIN IMAGING TRACERS FOR THE NORADRENALINE TRANSPORTER: In vivo imaging of the noradrenaline transporter was previously limited to peripheral tracers such as metaiodobenzylguanidine (MIBG), which does not cross the blood-brain barrier, and neuroligands such as radiolabelled desiprimine, which demonstrated problematically high nonspecific binding. Recent efforts in the literature have focused upon modified reboxetine analogues, and some progress was made with the S,S-isomer of iodophenoxy-ring reboxetine, referred to as both INER and IPBM in publication (Tamagnan et al. 2007; Kanegawa et al. 2006). Synthesised iodophenyl-ring compounds in S,S-, R,R-, S,R-, and R,S-isomers were evaluated for their affinity for the NAT. The R,S-isomer demonstrated a very similar level of binding to the S,S-isomer and so this was explored in an iodophenoxy isomer to determine if improvement upon the characteristics of lead iodinated reboxetine compounds in the literature was possible. The R,S-isomer iodophenoxy reboxetine analogues were synthesised with the iodine in the ortho, meta, and para positions of the phenoxy ring and assigned the designations NKJ-64, NKJ-67, and NKJ-68, respectively. NKJ-64 has strong affinity and selectivity for the noradrenaline transporter, with a KD of 8.4 ± 1.7 nM, a 6-fold selectivity for the noradrenaline transporter over the serotonin transporter, and a 63-fold selectivity for the noradrenaline transporter over the dopamine transporter. From the compounds synthesised and tested, NKJ-64 has the most suitable pharmacology to be developed further as a SPECT brain imaging tracer for the noradrenaline transporter and has an affinity in the same order of magnitude as iodinated compounds in the literature, such as the aforementioned INER/IPBM. USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) TO PREDICT IN VIVO CHARACTERISTICS OF TRACER CANDIDATES: The development of any candidate tracer compound must include an evaluation of its in vivo properties. To streamline this process and select only the most viable compounds for further testing, predictors of in vivo molecular characteristics are used to conserve research effort. Measures such as lipophilicity, phospholipophilicity, and plasma protein binding, used to estimate the potential for blood-brain barrier penetration, nonspecific binding, and the likelihood of serum availability would be very time-intensive to determine via traditional bench-top methodologies. High-performance liquid chromatography (HPLC) is a quick, precise, and reliable technique that is a rapid and high-throughput automatable process with exceptional precision that does not rely on the bench-top proficiency of an individual experimenter. The optimisation of gradient elution protocols and formulae to interpret sample retention times on commercially available columns into reproducible standardised units has allowed for the efficient evaluation of tracer candidate compounds. The iodinated reboxetine compounds were compared to examples of both successful and failed neuroimaging tracers. Generally, the evaluations of the successful and failed neuroimaging tracers validated the combined methodologies of the HPLC-derived predictors of in vivo molecular behaviour. In applying these methodologies to the synthesised iodoreboxetine compounds, NKJ-64 displayed many of the HPLC-determined properties that are consistent with successful tracers

The endocannabinoid system in experimental models of Alzheimer's disease

415 p.Durante la progresión de la enfermedad de Alzheimer (EA) se produce una degeneración del sistema de neurotransmisión colinérgico del prosencéfalo basal, que conduce a una pérdida progresiva e irreversible de funciones cognitivas, así como alteraciones del sistema endocannabinoide (eCB) y cambios en la composición lipídica cerebral. Cada vez más estudios apuntan al posible papel neuroprotector del sistema eCB en procesos neurodegenerativos y ya han sido demostrados los efectos beneficiosos de agonistas cannabinoides para el tratamiento de alteraciones conductuales asociadas a la EA. El sistema eCB modula entre otros al sistema colinérgico a través del receptor CB1 y, la síntesis de sus ligandos está ligada a la señalización muscarínica colinérgica en áreas que controlan procesos cognitivos. En este trabajo se ha estudiado el papel del sistema eCB en diferentes modelos de degeneración colinérgica y de EA. La inmunotoxina 192IgG-saporina elimina específicamente neuronas colinérgicas del prosencéfalo basal y se ha utilizado en cultivos organotípicos y ratas adultas como modelos ex vivo e in vivo de EA. Además se han administrado cannabinoides en el modelo 3xTg-AD para la EA y evaluado aprendizaje y memoria. El tratamiento con cannabinoides muestra protección sobre la muerte celular asociada a la lesión colinérgica en el modelo ex vivo. Los estudios in vivo, tras la administración de la inmunotoxina, muestran un deterioro cognitivo, una gran pérdida de inervación colinérgica cortical, un proceso adaptativo del sistema eCB a través del receptor CB1 y una profunda alteración del perfil fosfolipídico, todo ello asociado a la lesión colinérgica. El modelo 3xTg-AD presenta alteraciones conductuales asociadas a una desregulación del sistema eCB, que son parcialmente revertidas tras la activación subcrónica del receptor CB1. Proponemos al sistema eCB como modulador de la neurotransmisión colinérgica en neurodegeneración y potencial diana de intervención terapéutica en la EA

The endocannabinoid system in experimental models of Alzheimer's disease

415 p.Durante la progresión de la enfermedad de Alzheimer (EA) se produce una degeneración del sistema de neurotransmisión colinérgico del prosencéfalo basal, que conduce a una pérdida progresiva e irreversible de funciones cognitivas, así como alteraciones del sistema endocannabinoide (eCB) y cambios en la composición lipídica cerebral. Cada vez más estudios apuntan al posible papel neuroprotector del sistema eCB en procesos neurodegenerativos y ya han sido demostrados los efectos beneficiosos de agonistas cannabinoides para el tratamiento de alteraciones conductuales asociadas a la EA. El sistema eCB modula entre otros al sistema colinérgico a través del receptor CB1 y, la síntesis de sus ligandos está ligada a la señalización muscarínica colinérgica en áreas que controlan procesos cognitivos. En este trabajo se ha estudiado el papel del sistema eCB en diferentes modelos de degeneración colinérgica y de EA. La inmunotoxina 192IgG-saporina elimina específicamente neuronas colinérgicas del prosencéfalo basal y se ha utilizado en cultivos organotípicos y ratas adultas como modelos ex vivo e in vivo de EA. Además se han administrado cannabinoides en el modelo 3xTg-AD para la EA y evaluado aprendizaje y memoria. El tratamiento con cannabinoides muestra protección sobre la muerte celular asociada a la lesión colinérgica en el modelo ex vivo. Los estudios in vivo, tras la administración de la inmunotoxina, muestran un deterioro cognitivo, una gran pérdida de inervación colinérgica cortical, un proceso adaptativo del sistema eCB a través del receptor CB1 y una profunda alteración del perfil fosfolipídico, todo ello asociado a la lesión colinérgica. El modelo 3xTg-AD presenta alteraciones conductuales asociadas a una desregulación del sistema eCB, que son parcialmente revertidas tras la activación subcrónica del receptor CB1. Proponemos al sistema eCB como modulador de la neurotransmisión colinérgica en neurodegeneración y potencial diana de intervención terapéutica en la EA

Études de la subthalamotomie comme traitement des dyskinésies chez le primate parkinsonien

La présente thèse comprend une étude des mécanismes neurochimiques d’une approche chirurgicale, la subthalamotomie, pour le traitement de la maladie de Parkinson et les dyskinésies induites à la L-DOPA. Nous avons cherché à identifier, à l’aide de quelques hypothèses de recherche, les changements biochimiques dans les ganglions de la base induits par la lésion du noyau sous-thalamique. Nous avons utilisé un modèle de singe parkinsonien traité à la L-DOPA et ayant reçu une subthalamotomie unilatérale. Nos résultats démontrent que la subthalamotomie potentialise la réponse à faible dose de L-DOPA et que cette potentialisation serait entre autre régulée par le récepteur dopaminergique D1 et les récepteurs glutamatergiques métabotropiques. Ces données apportent de nouveaux éléments aidant à mieux comprendre les mécanismes de cette chirurgie pour le traitement des dyskinésies induites à la L-DOPA. De telles connaissances ouvrent la porte à de nouvelles stratégies pour augmenter la réponse chirurgicale du patient.Lesion of the subthalamic nucleus, also called subthalamotomy, is surgical therapy offered to parkinsonian patients refractory to L-DOPA or for whom L-DOPA-induced dyskinesias become disabling. Its mechanisms remain however largely unknown. In order to better understand the biochemical and cellular changes underlying subthalamotomy, we used an Parkinson’s disease animal model, the MPTP monkey. Chronic administration of L-DOPA in this animal model induces dyskinesias, as those seen in parkinsonian patients. The monkeys used in this study displayed such side effects and took part of different pharmacological trials to reduce these dyskinesias before undergoing surgery. Thus, we replicated the clinical situation where patients receive such surgery when all the other pharmacological treatments have failed. These monkeys received a unilateral subthalamotomy, the non-lesioned side served as an intra-animal control. Antiparkinsonian response to low dose of L-DOPA was potentiated by subthalamotomy. Then, we studied by autoradiography the D1 and D2 dopaminergic receptors, ionotropic NMDA (NR1/NR2B) and AMPA, metabotropic mGluR2/3 and mGluR5 glutamatergic receptors, and the dopaminergic transporter (DAT) using respectively the selective radioligands [3H]-SCH-23390, [3H]-Raclopride, [3H]-Ro 25-6981, [3H]-Ro 48-8587, [3H]-LY-341495, [3H]-ABP688 and [125I]-RTI-121. We measured by in situ hybridization the D1, D2 and preproenkephalin mRNAs using oligonucleotides as well as preprodynorphin mRNA using a riboprobe. We also assessed the dopamine and its metabolites by high-performance liquid chromatography. Finally, we measured the proteins ERK1 and ERK2, involved in intracellular signaling, and their respective phosphorylation state, as well as DARPP-32 by Western blot. Our results show that the dopamine D1 receptor, but not D2, as well as the metabotropic glutamate receptors are involved in the behavioral effects of subthalamotomy. This data suggest that the potentiation of response to L-DOPA after subthalamotomy would be due to changes in the direct pathway of the model of basal ganglia and in the subthalamic output. Our results open new and exciting pathways to explore on subthalamotomy, as well as other surgeries that are offered to disabled patients with movement disorders, whether these surgeries are lesional or with implantable stimulation devices