Molecular mechanisms involved in neurotoxicity mediated by HIV proteins and drug abuse

While the advancement of highly active antiretroviral therapy (HAART) has transformed the course of HIV/AIDS from a death sentence to a manageable chronic condition, the prevalence of a constellation of neurological disorders collectively termed as HIV-associated neurocognitive disorders (HAND) continues to persist in these patients. HAND is characterized by cognitive dysfunction, depression, impaired memory and/or deficits in motor skills. The underlying factors leading to HAND have been the subject of extensive research and are thought to be associated with HIV infection in the CNS combined with robust immune activation of resident cells of the CNS. In addition, there is a strong correlation between chronic substance abuse and the manifestation of HAND, posing a major challenge for health care management in HIV-positive drug abusers. One of the hallmark features of cocaine and opiate abuse is the increased neuronal toxicity in the setting of HIV infection. Among the various commonly abused drugs, cocaine has been extensively studied for its ability to exacerbate the neuropathogenesis of HAND. Ample evidence suggests that cocaine not only facilitates viral replication in astrocytes and microglia, enhances the permeability of the blood-brain barrier (BBB) and exacerbates neuroinflammatory responses, working synergistically with viral proteins such as HIV Tat and virus envelop protein Gp120 to promote neuronal injury. Sigma-1 receptors (Sig-1R) are recognized as a unique class of non-G protein-coupled intracellular protein that binds to their ligands such as cocaine, resulting in dissociation of Sig-1R from mitochondrion-associated ER membrane (MAM) to the endoplasmic reticulum (ER), plasma membrane, and nuclear membrane, regulating function of various proteins. Sig-1R has diverse roles in both physiological as well as in pathogenic processes. The disruption of Sig-1R pathways has been implicated as causative mechanism(s) in the development of neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and Huntington Disease (HD). Additionally, the interaction of cocaine and Sig-1R has more recently been implicated in potentiating the pathogenesis of HAND through impairment of BBB, microglial activation and astrogliosis. Opiates such as morphine are commonly used clinically in palliative care and pain management. Chronic exposure to morphine and other opioids however, can result in significant detrimental effects on cognition. Our findings suggest that morphine dysregulates synaptic balance in the hippocampus, a key center for learning and memory, via a novel signaling pathway involving reactive oxygen species (ROS), endoplasmic reticulum stress (ER stress) and autophagy. We demonstrate herein that morphine treatment leads to a reduction in excitatory and a concomitant enhancement of inhibitory synapse densities in the hippocampal neurons via activation of the mu opioid receptor. Furthermore, these effects are mediated by the ability of morphine to upregulate intracellular ROS from nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), which in turn, promotes ER stress and the induction of autophagy, leading ultimately to decreased excitatory and increased inhibitory synapse densities. The detrimental effects of morphine on synaptic densities were shown to be reversed by platelet-derived growth factor (PDGF), a pleiotropic growth factor, that has been implicated in neuroprotection, at the level of intracellular ROS. Both abnormal unfolded protein response (UPR) and impaired autophagy have also been implicated as a causative mechanism in the development of various neurodegenerative diseases. The common underlying feature of most neurodegenerative diseases such as AD, prion diseases, PD, and ALS involves accumulation of misfolded proteins leading to initiation of endoplasmic ER stress and stimulation of the UPR. Additionally, ER stress has more recently been implicated in the pathogenesis of HAND. Autophagy plays an essential role in the clearance of aggregated toxic proteins and degradation of the damaged organelles. These results thus identify a novel cellular mechanism involved in morphine-mediated synaptic alterations with implications for impaired cognition as well as neurocognitive disorders. Furthermore, based on our findings, it can be speculated that therapeutic strategies aimed at activating PDGF signaling can be envisioned as possible approaches to block morphine-mediated cognitive decline

The netrin receptor DCC is required in the pubertal organization of mesocortical dopamine circuitry

Netrins are guidance cues involvedinneural connectivity.Wehave shownthat the netrin-1 receptor DCC (deletedin colorectal cancer) is involvedinthefunctionalorganizationofthemesocorticolimbic dopamine(DA)system.Adult micewithaheterozygousloss-of-function mutation in dcc exhibit changes in indexes of DA function, including DA-related behaviors. These phenotypes are only observed after puberty,acritical periodinthe maturationofthe mesocortical DAprojection. Here, weexamined whether dcc heterozygous mice exhibit structural changes in medial prefrontal cortex (mPFC) DA synaptic connectivity, before and after puberty. Stereological counts of tyrosine-hydroxylase (TH)-positive varicosities were increased in the cingulate 1 and prelimbic regions of the pregenual mPFC. dcc heterozygous mice also exhibited alterations in the size, complexity, and dendritic spine density of mPFC layer V pyramidal neuron basilar dendritic arbors. Remarkably, these presynaptic and postsynaptic partner phenotypes were not observed in juvenile mice, suggesting that DCC selectively influences the extensive branching and synaptic differentiation that occurs in the maturing mPFC DA circuitatpuberty.Immunolabelingexperimentsinwild-typemice demonstratedthat DCCissegregatedtoTH-positivefibersinnervating the nucleus accumbens, with only scarce DCC labeling in mPFC TH-positive fibers. Netrin had an inverted target expression pattern. Thus, DCC-mediated netrin-1 signaling may influence the formation/maintenance of mesocorticolimbic DA topography. In support of this, we report that dcc heterozygous mice exhibit a twofold increase in the density of mPFC DCC/TH-positive varicosities. Our results implicate DCC-mediated netrin-1 signaling in the establishment of mPFC DA circuitry during puberty

Autoregulatory and Paracrine Control of Synaptic and Behavioral Plasticity by Dual Modes of Octopaminergic Signaling: A Dissertation

Synaptic plasticity—the ability of a synapse to change—is fundamental to basic brain function and behavioral adaptation. Studying the mechanisms of synaptic plasticity benefits our understanding of the formation of neuronal connections and circuitry, which has great implications in the field of learning and memory and the studies of numerous human diseases. The Drosophila larval neuromuscular junction (NMJ) system is a powerful system for studying synaptic plasticity. The NMJ consists of at least two different types of motorneurons innervating the body wall muscles. Type I motorneurons controls muscle contraction using glutamate as the neurotransmitter, while type II are modulatory neurons that contain octopamine. Octopamine is a potent modulator of behavior in invertebrates. Nevertheless, its function at the synapse is poorly understood. In my thesis research, I investigated the role of octopamine in synaptic plasticity using the Drosophila NMJ system. Preliminary observations indicate that increased larval locomotion during starvation results in an increase of filopodia-like structures at type II terminals. These structures, which we termed as “synaptopods” in our previous studies, contain synaptic proteins and can mature into type II synapses. I demonstrated that this outgrowth of type II terminals is dependent on activity and octopamine. Mutations and genetic manipulations affecting the production of octopamine decrease synaptopods, whereas increase of type II activity or exogenous application of octopamine increase synaptopods. Interestingly, I found that the type II octopaminergic neurons have an absolute dependence on activity for their innervation of the muscles. Blocking activity in these neurons throughout development results in no type II synapses at the NMJ, whereas blocking activity after the formation of synapses results in gradual degradation of type II terminals. Next, I examined the autoregulatory mechanism underlying the octopamine-induced synaptic growth in octopaminergic neurons. I discovered that this positive-feedback mechanism depends on an octopamine autoreceptor, Octß2R. This receptor in turn activates a cAMP- and CREB-dependent pathway that is required in the octopamine-induction of synaptopods. Furthermore, I demonstrated that this octopaminergic autoregulatory mechanism is necessary for the larva to properly increase its locomotor activity during starvation. Thirdly, I investigated the possibility that type II innervation might regulate type I synaptic growth through octopamine. We found that ablation, blocking of type II activity, or the absence of octopamine results in reduced type I outgrowth, and this paracrine signaling is mediated by Octß2R which is also present in type I motorneurons. Lastly, the function of another octopamine receptor, Octß1R, was examined. In contrast to Octß2R, Octß1R is inhibitory to synaptic growth. I demonstrated that the inhibitory effect of this receptor is likely accomplished through the inhibitory G-protein Goα. Similar to Octß2R, Octß1R also regulates the synaptic growth of both type I and type II motorneurons in a cell-autonomous manner. The inhibitory function of this receptor potentially breaks the positive feedback loop mediated by Octß2R, allowing the animal to reset its neurons when the environment is favorable. In summary, the research presented in this thesis has unraveled both autoregulatory and paracrine mechanisms in which octopamine modulates synaptic and behavior plasticity through excitatory and inhibitory receptors

Innervation cholinergique du cortex cérébral chez le rat adulte et en cours de développement : distribution quantifiée et analyse ultrastructurale

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Crossroads of Drug Abuse and HIV Infection: Neurotoxicity and CNS Reservoir

Drug abuse is a common comorbidity in people infected with HIV. HIV-infected individuals who abuse drugs are a key population who frequently experience suboptimal outcomes along the HIV continuum of care. A modest proportion of HIV-infected individuals develop HIV-associated neurocognitive issues, the severity of which further increases with drug abuse. Moreover, the tendency of the virus to go into latency in certain cellular reservoirs again complicates the elimination of HIV and HIV-associated illnesses. Antiretroviral therapy (ART) successfully decreased the overall viral load in infected people, yet it does not effectively eliminate the virus from all latent reservoirs. Although ART increased the life expectancy of infected individuals, it showed inconsistent improvement in CNS functioning, thus decreasing the quality of life. Research efforts have been dedicated to identifying common mechanisms through which HIV and drug abuse lead to neurotoxicity and CNS dysfunction. Therefore, in order to develop an effective treatment regimen to treat neurocognitive and related symptoms in HIV-infected patients, it is crucial to understand the involved mechanisms of neurotoxicity. Eventually, those mechanisms could lead the way to design and develop novel therapeutic strategies addressing both CNS HIV reservoir and illicit drug use by HIV patients

Molecular fingerprinting of VNUT-containing compartments in Neuro-2a cells

Vesicular ATP release is involved in regulating biological processes like nociception, blood glucose, and vascular tone. ATP-containing vesicles are filled by the vesicular nucleotide transporter (VNUT) and are molecularly distinct from catecholaminergic vesicles of sympathetic neurons. This work sought to identify the molecular fingerprint of VNUT-containing vesicles. Fluorescence microscopy in Neuro-2a, HeLa, and HEK293 cells showed VNUT being widely dispersed throughout the cells with a perinuclear enrichment. VNUT failed to colocalize with known markers of synaptic vesicles, lysosomes, dense cored vesicles, and catecholaminergic vesicles. Bioinformatic analyses of mammalian VNUT C-terminus identified a unique KDEL-like HEDL motif, as well as a lack of classic synaptic vesicle-targeting dileucine-like and tyrosine-based motifs. This work suggests that VNUT likely resides primarily in the Golgi-ER complex, a previously unconsidered location for what is thought to be a vesicle-associated translocase

Implication de collatérales axonales locales dans la libération de dopamine dans le mésencéphale

Les neurones dopaminergiques (DAergiques) libèrent non seulement de la DA à partir de leurs terminaisons axonales, mais également dans le mésencéphale au niveau de la substance noire (SN) et l’aire tegmentaire ventrale (ATV). À cet endroit, un mécanisme de libération somatodendritique (STD) de DA a été proposé et impliquerait des senseurs calciques différents de ceux retrouvés du côté axonal. Au niveau axonal, la synaptotagmine 1 (Syt1) est une protéine essentielle à la libération rapide de DA. Toutefois, des études de notre laboratoire sur des knockout conditionnels (cKO) de Syt1 dans les neurones DA démontrent une diminution substantielle de la libération de DA au niveau axonal, mais aussi dans le mésencéphale. Une première hypothèse expliquant cette diminution dans le mésencéphale serait que Syt1 est impliquée dans la libération STD. Cependant, nous observons par microscopie à super-résolution que Syt1 ne se retrouve pas dans le compartiment STD des neurones DAergiques. Une autre possibilité serait la présence de collatérales axonales DAergiques dans le mésencéphale. Par imagerie confocale et électronique, nous observons que le mésencéphale contient plusieurs varicosités axonales asynaptiques et quelques varicosités axonales synaptiques. Enfin, nous avons évalué la plasticité des collatérales axonales DAergiques dans un modèle de lésion partielle des neurones DAergiques induite par la 6-OHDA. Malgré la perte de plus de 40% des neurones DA, la libération de DA dans la SN persiste 14 jours après lésion et s’accompagne d’une augmentation de l’expression axonale de Syt1, suggérant qu’un mécanisme de compensation axonale contribue à la résilience de la libération de DA.Dopaminergic (DA) neurons not only exhibit a classic vesicular release from their axons in the striatum, but they also release DA in the midbrain in the substantia nigra (SN) and ventral tegmental area (VTA). In this region, somatodendritic (STD) release occurs and it requires different calcium sensors than those found in the axons. Of interest, synaptotagmin 1 (Syt1) has been shown to be implicated in fast DA release in the axons. However, recent research in our lab shows that in mice with conditional knockout (cKO) of Syt1 in DA neurons, there is a substantial decrease of DA release not only in the striatum, but also in the midbrain. Our first hypothesis is that Syt1 is directly involved in STD release. With super-resolution microscopy, we concluded that Syt1 is not localized in the STD compartment of DA neurons. This brings us to our second hypothesis, where local DA axon collaterals contribute to DA release in the midbrain. Through confocal and electron microscopy, we observed that the midbrain contains asynaptic varicosities as well as local DA synapses. In light of these results, we explored the contribution of axonal release to the resilience of SN DA release in a partial 6-OHDA lesion model. We observed that, following a loss of more than 40% of DA neurons, DA release in the SNc persists 14 days after lesion and that this is accompanied by an increase in Syt1 expression in DA axons which suggest that local axonal release is increased to compensate for DA loss