Sleep-controlling neurons are sensitive and vulnerable to multiple forms of α-synuclein: implications for the early appearance of sleeping disorders in α-synucleinopathies.

Parkinson's disease, Multiple System Atrophy, and Lewy Body Dementia are incurable diseases called α-synucleinopathies as they are mechanistically linked to the protein, α-synuclein (α-syn). α-syn exists in different structural forms which have been linked to clinical disease distinctions. However, sleeping disorders (SDs) are common in the prodromal phase of all three α-synucleinopathies, which suggests that sleep-controlling neurons are affected by multiple forms of α-syn. To determine whether a structure-independent neuronal impact of α-syn exists, we compared and contrasted the cellular effect of three different α-syn forms on neurotransmitter-defined cells of two sleep-controlling nuclei located in the brainstem: the laterodorsal tegmental nucleus and the pedunculopontine tegmental nucleus. We utilized size exclusion chromatography, fluorescence spectroscopy, circular dichroism spectroscopy and transmission electron microscopy to precisely characterize ​​timepoints in the α-syn aggregation process with three different dominating forms of this protein (monomeric, oligomeric and fibril) and we conducted an in-depth investigation of the underlying neuronal mechanism behind cellular effects of the different forms of the protein using electrophysiology, multiple-cell calcium imaging, single-cell calcium imaging and live-location tracking with fluorescently-tagged α-syn. Interestingly, α-syn altered membrane currents, enhanced firing, increased intracellular calcium and facilitated cell death in a structure-independent manner in sleep-controlling nuclei, and postsynaptic actions involved a G-protein-mediated mechanism. These data are novel as the sleep-controlling nuclei are the first brain regions reported to be affected by α-syn in this structure-independent manner. These regions may represent highly important targets for future neuroprotective therapy to modify or delay disease progression in α-synucleinopathies.

α-Synuclein Responses in the Laterodorsal Tegmentum, the Pedunculopontine Tegmentum, and the Substantia Nigra: Implications for Early Appearance of Sleep Disorders in Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder associated with insoluble pathological aggregates of the protein α-synuclein. While PD is diagnosed by motor symptoms putatively due to aggregated α-synuclein-mediated damage to substantia nigra (SN) neurons, up to a decade before motor symptom appearance, patients exhibit sleep disorders (SDs). Therefore, we hypothesized that α-synuclein, which can be present in monomeric, fibril, and other forms, has deleterious cellular actions on sleep-control nuclei. OBJECTIVE: We investigated whether native monomer and fibril forms of α-synuclein have effects on neuronal function, calcium dynamics, and cell-death-induction in two sleep-controlling nuclei: the laterodorsal tegmentum (LDT), and the pedunculopontine tegmentum (PPT), as well as the motor-controlling SN. METHODS: Size exclusion chromatography, Thioflavin T fluorescence assays, and circular dichroism spectroscopy were used to isolate structurally defined forms of recombinant, human α-synuclein. Neuronal and viability effects of characterized monomeric and fibril forms of α-synuclein were determined on LDT, PPT, and SN neurons using electrophysiology, calcium imaging, and neurotoxicity assays. RESULTS: In LDT and PPT neurons, both forms of α-synuclein induced excitation and increased calcium, and the monomeric form heightened putatively excitotoxic neuronal death, whereas, in the SN, we saw inhibition, decreased intracellular calcium, and monomeric α-synuclein was not associated with heightened cell death. CONCLUSION: Nucleus-specific differential effects suggest mechanistic underpinnings of SDs' prodromal appearance in PD. While speculative, we hypothesize that the monomeric form of α-synuclein compromises functionality of sleep-control neurons, leading to the presence of SDs decades prior to motor dysfunction.

Characterization of AN317, a novel selective agonist of α6ß2-containing nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are crucial mediators of central presynaptic, postsynaptic, and extrasynaptic signaling, and they are implicated in a range of CNS disorders. The numerous nAChR subtypes are differentially expressed and mediate distinct functions throughout the CNS, and thus there is considerable interest in developing subtype-selective nAChR modulators, both for use as pharmacological tools and as putative therapeutics. α6ß2-containing (α6ß2*) nAChRs are highly expressed in and regulate the activity of midbrain dopaminergic neurons, which makes them attractive drug targets in several psychiatric and neurological diseases, including nicotine addiction and Parkinson's disease. This paper presents the preclinical characterization of AN317, a novel α6ß2* agonist exhibiting functional selectivity toward other nAChRs, including α4ß2, α3ß4 and α7 receptors. AN317 induced [3H]dopamine release from rat striatal synaptosomes and augmented dopaminergic neuron activity in substantia nigra pars compacta brain slices in Ca2+ imaging and electrophysiological assays. In line with this, AN317 alleviated the high-frequency tremors arising from reserpine-mediated dopamine depletion in rats. Finally, AN317 mediated significant protective effects on cultured rat mesencephalic neurons treated with the dopaminergic neurotoxin MPP+. AN317 displays good bioavailability and readily crosses the blood-brain barrier, which makes it a unique tool for both in vitro and in vivo studies of native α6ß2* receptors in the nigrostriatal system and other dopaminergic pathways. Altogether, these findings highlight the potential of selective α6ß2* nAChR activation as a treatment strategy for symptoms and possibly even deceleration of disease progression in neurodegenerative diseases such as Parkinson's disease.

Characterization of AN6001, a positive allosteric modulator of α6ß2-containing nicotinic acetylcholine receptors.

α6ß2-Containing nicotinic acetylcholine receptors (α6ß2* nAChRs) are predominantly expressed in midbrain dopaminergic neurons, including substantia nigra pars compacta (SNc) neurons and their projections to striatal regions, where they regulate dopamine release and nigrostriatal activity. It is well established that nAChR agonists exert protection against dopaminergic neurotoxicity in cellular assays and parkinsonian animal models. Historically, drug development in the nAChR field has been mostly focused on development of selective agonists and positive allosteric modulators (PAMs) for the predominant neuronal nAChRs, α7 and α4ß2. Here, we report the discovery and characterization of AN6001, a novel selective α6ß2* nAChR PAM. AN6001 mediated increases in both nicotine potency and efficacy at the human α6/α3ß2ß3V9'S nAChR in HEK293 cells, and it positively modulated ACh-evoked currents through both α6/α3ß2ß3V9'S and a concatenated ß3-α6-ß2-α6-ß2 receptor in Xenopus oocytes, displaying EC50 values of 0.58 µM and 0.40 µM, respectively. In contrast, the compound did not display significant modulatory activity at α4ß2, α3ß4, α7 and muscle nAChRs. AN6001 also increased agonist-induced dopamine release from striatal synaptosomes and augmented agonist-induced global cellular responses and inward currents in dopaminergic neurons in SNc slices (measured by Ca2+ imaging and patch clamp recordings, respectively). Finally, AN6001 potentiated the neuroprotective effect of nicotine at MPP+-treated primary dopaminergic neurons. Overall, our studies demonstrate the existence of allosteric sites on α6ß2* nAChRs and that positive modulation of native α6ß2* receptors strengthens DA signaling. Hence, AN6001 represents an important tool for studies of α6ß2* nAChRs and furthermore underlines the therapeutic potential in these receptors in Parkinson's disease.

Hair in Parkinson's disease patients exhibits differences in Calcium, Iron and Zinc concentrations measured by flame atomic absorption spectrometry - FAAS.

Imbalances in metals have emerged as playing a role in the pathophysiology of Parkinson's Disease (PD). Monitoring of metal levels could serve as a biomarker of presence, or future development, of this disease. To this end, we evaluated the ability of flame atomic absorption spectrometry (FAAS) to assess the concentrations of Ca, Fe and Zn in hair of PD patients and to investigate if there was an association with age and disease duration. Hair samples were collected from 26 clinically-diagnosed PD patients, and 33 healthy individuals. Concentrations of Ca and Fe were lower in PD patients when compared to control, whereas, a higher concentration of Zn was detected in PD patients. Levels of Ca and Fe did not vary with age nor with the duration of PD. While Zn did not present variation with duration of the disease, there was a correlation with age as PD patients older than 65 years exhibited a higher concentration of Zn than controls. We conclude that FAAS is useful for detecting differences in Fe, Ca and Zn in hair samples of patients with PD. Hair samples required for this method are easy to collect, and the technique relies on a simple method of digestion of the organic matrix. The ease of use of FAAS should allow for more frequent monitoring of metallic levels in patients in a variety of small clinical situations, thereby offering the hope of allowing systematic tracking of metal levels as the disease progresses, or prior to the defining motor symptoms.

Growth Factors and Astrocytes Metabolism: Possible Roles for Platelet Derived Growth Factor.

Astrocytes exert multiple functions in the brain such as the development of blood-brain barrier characteristics, the promotion of neurovascular coupling, attraction of cells through the release of chemokines, clearance of toxic substances and generation of antioxidant molecules and growth factors. In this aspect, astrocytes secrete several growth factors (BDNF, GDNF, NGF, and others) that are fundamental for cell viability, oxidant protection, genetic expression and modulation of metabolic functions. The platelet derived growth factor (PDGF), which is expressed by many SNC cells, including astrocytes, is an important molecule that has shown neuroprotective potential, improvement of wound healing, regulation of calcium metabolism and mitochondrial function. Here we explore some of these astrocyte-driven functions of growth factors and their possible therapeutic uses in the context of neurodegeneration.

Are sleep disturbances preclinical markers of Parkinson's disease?

Parkinson's disease (PD) is a neurobehavioral disorder characterized by motor symptoms and signs, and non-motor abnormalities such as olfactory dysfunction, pain, sleep disorders and cognitive impairment. Amongst these alterations, sleep disturbances play an important role in the pathology, but presence of disturbed sleep is not currently considered in diagnosis. However, sleeping problems may precede by many years the classic motor abnormalities of PD and should be clinically evaluated as a potential marker before disease onset. The first disturbance reported with this potential was the disorder REM sleep behaviour and currently several other disturbances have gained importance as potential markers, such as excessive daytime sleepiness, restless legs syndrome and new evidence also points to changes in circadian rhythms. Here we present a brief review of the major evidence indicating that sleep disturbances precede the motor symptoms in PD and neurodegeneration occurs in regions that could underlie these phenomena in order to provide support for the conclusion that disturbances of sleep should be considered as valuable preclinical markers for PD.