Effects of Neurotrophic Factors in Glial Cells in the Central Nervous System : Expression and Properties in Neurodegeneration and Injury

Astrocytes, oligodendrocytes, and microglia are abundant cell types found in the central nervous system and have been shown to play crucial roles in regulating both normal and disease states. An increasing amount of evidence points to the critical importance of glia in mediating neurodegeneration in Alzheimer's and Parkinson's diseases (AD, PD), and in ischemic stroke, where microglia are involved in initial tissue clearance, and astrocytes in the subsequent formation of a glial scar. The importance of these cells for neuronal survival has previously been studied in co-culture experiments and the search for neurotrophic factors (NTFs) initiated after finding that the addition of conditioned media from astrocyte cultures could support the survival of primary neurons in vitro. This led to the discovery of the potent dopamine neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF). In this review, we focus on the relationship between glia and NTFs including neurotrophins, GDNF-family ligands, CNTF family, and CDNF/MANF-family proteins. We describe their expression in astrocytes, oligodendrocytes and their precursors (NG2-positive cells, OPCs), and microglia during development and in the adult brain. Furthermore, we review existing data on the glial phenotypes of NTF knockout mice and follow NTF expression patterns and their effects on glia in disease models such as AD, PD, stroke, and retinal degeneration.Peer reviewe

GDNF/RET signalling in regulation of brain dopaminergic systems : significance for drug addiction

Glial cell line-derived neurotrophic factor (GDNF) has been shown to be a potent neurotrophic factor for the brain dopaminergic systems. GDNF has been shown to protect and promote recovery of brain dopaminergic neurons in animal models of Parkinson's disease. As brain dopaminergic pathways are important for perceiving drug reward, and as they undergo plastic changes during chronic drug use, interactions between GDNF and responses to abused drugs are of interest. This study investigated the role of endogenous GDNF in the regulation of nigrostriatal and mesolimbic dopaminergic neurotransmission. The effects of acutely and repeatedly administered cocaine and morphine on striatal dopamine release, on behavioural sensitization and on drug reward were studied in heterozygous GDNF knockout mice. In addition, this study explored the effects of constitutive RET tyrosine kinase receptor activity on brain dopaminergic systems and on the locomotor enhancing effects of cocaine in mice. Unexpectedly, it was found that extracellular dopamine concentrations were increased in striatal brain areas in heterozygous GDNF knockout mice. This was further supported by a clear increase in the number of FosB/deltaFosB positive nuclei in the caudate/putamen and nucleus accumbens in these mice. Thus, the present results indicate that dopaminergic transmission is increased in mice with reduced GDNF levels. In addition, heterozygous GDNF+/- mice were more sensitive to morphine's dopamine releasing effect, and reduced GDNF levels were associated with a shift in the bell-shaped dose-response curve of morphine to the left. Interestingly, it was found that after repeated morphine and cocaine treatment, the extracellular dopamine concentrations in the GDNF+/- mice were decreased to a level similar to their wild-type littermates. In addition, it was found that reduced GDNF levels are involved in a more rapid development of tolerance to locomotor enhancing effects of daily 30 mg/kg morphine injections, and in increased sensitivity to locomotor sensitization by a 5 mg/kg morphine challenge dose. Furthermore, the present results show that constitutive RET activity, caused by a single point mutation Met919Thr, robustly increased dopamine concentrations in the brain, whereas noradrenaline or serotonin concentrations were not affected. Increased dopamine concentrations were associated with increased tyrosine hydroxylase protein levels, indicating that dopamine synthesis is increased in these mice. An important finding was that increased RET activity increased the number of nigrostriatal dopamine neurons in the adult mice. All in all, the present findings emphasize the important role of GDNF/RET-signalling in the regulation of brain dopaminergic systems.Gliasolulinjaperäisen hermokasvutekijän (GDNF) on osoitettu olevan tärkeä keskiaivojen dopaminergisten hermosolujen toiminnan säätelyssä. GDNF on laajalti tutkittu Parkinsonin taudin eläinmalleissa, ja sillä on havaittu olevan sekä dopaminergisiä hermosoluja suojaava että niiden toimintaa elvyttävä vaikutus. Koska huumeiden riippuvuutta aiheuttavat vaikutukset välittyvät dopaminergisten järjestelmien kautta, on hermokasvutekijöiden, ja erityisesti GDNF:n merkitys päihteiden aiheuttamissa keskushermostomuutoksissa mielenkiintoinen tutkimuksen kohde. Väitöskirjassani tutkittiin miten GDNF:n pitoisuuden vähentäminen aivoissa vaikuttaa dopaminergisen järjestelmän toimintaan. Tutkimuksessa selvitettiin kokaiinin ja morfiinin vaikutuksia dopamiinin vapautumiseen, liikeaktiivisuuden herkistymiseen ja palkitseviin vaikutuksiin käyttäen heterotsygootteja GDNF+/- poistogeenisiä hiiriä. Lisäksi tutkittiin miten GDNF:n vaikutuksia soluun välittävän reseptorin, RET tyrosiinikinaasireseptorin, aktiivisuuden lisääminen vaikuttaa aivojen dopaminergisiin järjestelmiin ja kokaiinin liikeaktiivisuutta lisääviin vaikutuksiin. Yllättäen havaittiin, että solunulkoinen dopamiinipitoisuus on lisääntynyt GDNF+/- hiirien striatumissa. Lisäksi havaittiin, että FosB/deltaFosB positiivisten tumien määrä oli lisääntynyt dopaminergisillä aivoalueilla GDNF+/- hiirissä. Tulos on mielenkiintoinen, koska deltaFosB-proteiinia voidaan pitää eräänlaisena riippuvuuden muistijälkenä. Nämä tulokset viittaavat siihen, että dopaminerginen neurotransmissio on näissä hiirissä lisääntynyt. Akuutisti annetun morfiinin havaittiin lisäävän dopamiinin vapautumista herkemmin GDNF+/- hiirissä kuin villityypin hiirissä. Lisäksi hiirissä, joissa aivojen GDNF-pitoisuus oli alentunut, havaittiin nopeampi toleranssin muodostuminen morfiinin liikeaktiivisuutta lisäävälle vaikutukselle, ja vierotuksen jälkeen nämä hiiret olivat herkempiä morfiinin liikeaktiivisuutta herkistävälle vaikutukselle. Lisäksi havaittiin, että kohonneet solunulkoiset dopamiinipitoisuudet laskivat GDNF+/- hiirissä villityyppi hiirien tasolle toistetun morfiini ja kokaiini annostelun jälkeen. Hiirissä, joilla RET aktiivisuus oli lisääntynyt, havaittiin merkittävästi suuremmat dopamiinipitoisuudet aivoissa post mortem, joka johtuu suuremmasta tyrosiinihydroksylaasi-proteiinin määrästä sekä osittain suuremmasta dopaminergisten solujen määrästä substantia nigra pars compactassa. Yhteenvetona havaintomme osoittavat, että GDNF/RET -signaloinnilla on tärkeä merkitys aivojen dopaminergisen järjestelmän säätelyssä

Neuroprotective and reparative effects of endoplasmic reticulum luminal proteins - mesencephalic astrocyte-derived neurotrophic factor and cerebral dopamine neurotrophic factor

Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are proteins that have received increasing attention in the last decades. Although they are called neurotrophic factors they are drastically different from neurotrophic factors in their expression and physiological actions.They are located in the lumen of the endoplasmic reticulum (ER) and their basal secretion from neurons is very low. However their secretion is stimulated upon ER calcium depletion by chemical probes such as thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor. Exogenous MANF and CDNF possess therapeutic properties in several neurological dkodse models, including Parkinson disease and stroke. Endogenoes MANF expression has been shown to be neuroprotective, as well as administration of either CDNF or MANF into the extracellular space. In this review, we focus on their therapeutic effects, regulation of expression and secretion, comparison of their mechanisms of action, and their application to the brain parenchyma as recombinant proteins.Peer reviewe

Morphological Heterogeneity of the Endoplasmic Reticulum within Neurons and Its Implications in Neurodegeneration

The endoplasmic reticulum (ER) is a multipurpose organelle comprising dynamic structural subdomains, such as ER sheets and tubules, serving to maintain protein, calcium, and lipid homeostasis. In neurons, the single ER is compartmentalized with a careful segregation of the structural subdomains in somatic and neurite (axodendritic) regions. The distribution and arrangement of these ER subdomains varies between different neuronal types. Mutations in ER membrane shaping proteins and morphological changes in the ER are associated with various neurodegenerative diseases implying significance of ER morphology in maintaining neuronal integrity. Specific neurons, such as the highly arborized dopaminergic neurons, are prone to stress and neurodegeneration. Differences in morphology and functionality of ER between the neurons may account for their varied sensitivity to stress and neurodegenerative changes. In this review, we explore the neuronal ER and discuss its distinct morphological attributes and specific functions. We hypothesize that morphological heterogeneity of the ER in neurons is an important factor that accounts for their selective susceptibility to neurodegeneration.Peer reviewe

AAV Vector-Mediated Gene Delivery to Substantia Nigra Dopamine Neurons : Implications for Gene Therapy and Disease Models

Gene delivery using adeno-associated virus (AAV) vectors is a widely used method to transduce neurons in the brain, especially due to its safety, efficacy, and long-lasting expression. In addition, by varying AAV serotype, promotor, and titer, it is possible to affect the cell specificity of expression or the expression levels of the protein of interest. Dopamine neurons in the substantia nigra projecting to the striatum, comprising the nigrostriatal pathway, are involved in movement control and degenerate in Parkinson's disease. AAV-based gene targeting to the projection area of these neurons in the striatum has been studied extensively to induce the production of neurotrophic factors for disease-modifying therapies for Parkinson's disease. Much less emphasis has been put on AAV-based gene therapy targeting dopamine neurons in substantia nigra. We will review the literature related to targeting striatum and/or substantia nigra dopamine neurons using AAVs in order to express neuroprotective and neurorestorative molecules, as well as produce animal disease models of Parkinson's disease. We discuss difficulties in targeting substantia nigra dopamine neurons and their vulnerability to stress in general. Therefore, choosing a proper control for experimental work is not trivial. Since the axons along the nigrostriatal tract are the first to degenerate in Parkinson's disease, the location to deliver the therapy must be carefully considered. We also review studies using AAV--synuclein (-syn) to target substantia nigra dopamine neurons to produce an -syn overexpression disease model in rats. Though these studies are able to produce mild dopamine system degeneration in the striatum and substantia nigra and some behavioural effects, there are studies pointing to the toxicity of AAV-carrying green fluorescent protein (GFP), which is often used as a control. Therefore, we discuss the potential difficulties in overexpressing proteins in general in the substantia nigra.Peer reviewe

Domain-Independent Inhibition of CBP/p300 Attenuates alpha-Synuclein Aggregation

Neurodegenerative diseases are associated with failed proteostasis and accumulation of insoluble protein aggregates that compromise neuronal function and survival. In Parkinson's disease, a major pathological finding is Lewy bodies and neurites that are mainly composed of phosphorylated and aggregated alpha-synuclein and fragments of organelle membranes. Here, we analyzed a series of selective inhibitors acting on multidomain proteins CBP and p300 that contain both lysine acetyltransferase and bromodomains and are responsible for the recognition and enzymatic modification of lysine residues. By using high-affinity inhibitors, A-485, GNE-049, and SGC-CBP30, we explored the role of two closely related proteins, CBP and p300, as promising targets for selective attenuation of alpha-synuclein aggregation. Our data show that selective CBP/p300 inhibitors may alter the course of pathological alpha-synuclein accumulation in primary mouse embryonic dopaminergic neurons. Hence, drug-like CBP/p300 inhibitors provide an effective approach for the development of high-affinity drug candidates preventing alpha-synuclein aggregation via systemic administration.Peer reviewe

Studying Pre-formed Fibril Induced α-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of α-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on α-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson’s disease, Lewy bodies (LB), is mimicked by the addition of α-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated α-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing α-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-α-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated α-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of α-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.Peer reviewe

Cerebral Dopamine Neurotrophic Factor Diffuses Around the Brainstem and Does Not Undergo Anterograde Transport After Injection to the Substantia Nigra

Cerebral dopamine neurotrophic factor (CDNF) has shown therapeutic potential in rodent and non-human primate models of Parkinson's disease by protecting the dopamine neurons from degeneration and even restoring their phenotype and function. Previously, neurorestorative efficacy of CDNF in the 6-hydroxydopamine (6-OHDA) model of Parkinson's disease as well as diffusion of the protein in the striatum (STR) has been demonstrated and studied. Here, experiments were performed to characterize the diffusion and transport of supra-nigral CDNF in non-lesioned rats. We injected recombinant human CDNF to the substantia nigra (SN) of naive male Wistar rats and analyzed the brains 2, 6, and 24 h after injections. We performed immunohistochemical stainings using an antibody specific to human CDNF and radioactivity measurements after injecting iodinated CDNF. Unlike the previously reported striatonigral retrograde transport seen after striatal injection, active anterograde transport of CDNF to the STR could not be detected after nigral injection. There was, however, clear diffusion of CDNF to the brain areas surrounding the SN, and CDNF colocalized with tyrosine hydroxylase (TH)-positive neurons. Overall, our results provide insight on how CDNF injected to the SN may act in this region of the brain.Peer reviewe

Back and to the Future: From Neurotoxin-Induced to Human Parkinson's Disease Models

Abstract Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by motor symptoms such as tremor, slowness of movement, rigidity, and postural instability, as well as non-motor features like sleep disturbances, loss of ability to smell, depression, constipation, and pain. Motor symptoms are caused by depletion of dopamine in the striatum due to the progressive loss of dopamine neurons in the substantia nigra pars compacta. Approximately 10% of PD cases are familial arising from genetic mutations in α-synuclein, LRRK2, DJ-1, PINK1, parkin, and several other proteins. The majority of PD cases are, however, idiopathic, i.e., having no clear etiology. PD is characterized by progressive accumulation of insoluble inclusions, known as Lewy bodies, mostly composed of α-synuclein and membrane components. The cause of PD is currently attributed to cellular proteostasis deregulation and mitochondrial dysfunction, which are likely interdependent. In addition, neuroinflammation is present in brains of PD patients, but whether it is the cause or consequence of neurodegeneration remains to be studied. Rodents do not develop PD or PD-like motor symptoms spontaneously; however, neurotoxins, genetic mutations, viral vector-mediated transgene expression and, recently, injections of misfolded α-synuclein have been successfully utilized to model certain aspects of the disease. Here, we critically review the advantages and drawbacks of rodent PD models and discuss approaches to advance pre-clinical PD research towards successful disease-modifying therapy. ? 2020 The Authors.Peer reviewe

Cell Culture Media, Unlike the Presence of Insulin, Affect α-Synuclein Aggregation in Dopaminergic Neurons

There are several links between insulin resistance and neurodegenerative disorders such as Parkinson’s disease. However, the direct influence of insulin signaling on abnormal α-synuclein accumulation—a hallmark of Parkinson’s disease—remains poorly explored. To our best knowledge, this work is the first attempt to investigate the direct effects of insulin signaling on pathological α-synuclein accumulation induced by the addition of α-synuclein preformed fibrils in primary dopaminergic neurons. We found that modifying insulin signaling through (1) insulin receptor inhibitor GSK1904529A, (2) SHIP2 inhibitor AS1949490 or (3) PTEN inhibitor VO-OHpic failed to significantly affect α-synuclein aggregation in dopaminergic neurons, in contrast to the aggregation-reducing effects observed after the addition of glial cell line-derived neurotrophic factor. Subsequently, we tested different media formulations, with and without insulin. Again, removal of insulin from cell culturing media showed no effect on α-synuclein accumulation. We observed, however, a reduced α-synuclein aggregation in neurons cultured in neurobasal medium with a B27 supplement, regardless of the presence of insulin, in contrast to DMEM/F12 medium with an N2 supplement. The effects of culture conditions were present only in dopaminergic but not in primary cortical or hippocampal cells, indicating the unique sensitivity of the former. Altogether, our data contravene the direct involvement of insulin signaling in the modulation of α-synuclein aggregation in dopamine neurons. Moreover, we show that the choice of culturing media can significantly affect preformed fibril-induced α-synuclein phosphorylation in a primary dopaminergic cell culture