The Cerebellum (2019) 18:1017–1035 https://doi.org/10.1007/s12311-019-01046-0

REVIEW

The Role of Astrocytes in the Development of the Cerebellum

Ana Paula Bergamo Araujo1 & Raul Carpi-Santos1 & Flávia Carvalho Alcantara Gomes1

Published online: 19 June 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract Astrocytes, initially described as merely support cells, are now known as a heterogeneous population of cells actively involved in a variety of biological functions such as: neuronal migration and differentiation; regulation of cerebral blood flow; metabolic control of extracellular potassium concentration; and modulation of synapse formation and elimination; among others. Cerebellar glial cells have been shown to play a significant role in proliferation, differentiation, migration, and synaptogenesis. However, less evidence is available about the role of neuron-astrocyte interactions during cerebellar development and their impact on diseases of the cerebellum. In this review, we will focus on the mechanisms underlying cellular interactions, specifically neuron- astrocyte interactions, during cerebellar development, function, and disease. We will discuss how cerebellar glia, astrocytes, and Bergmann glia play a fundamental role in several steps of cerebellar development, such as granule cell migration, axonal growth, neuronal differentiation, and synapse formation, and in diseases associated with the cerebellum. We will focus on how astrocytes and thyroid hormones impact cerebellar development. Furthermore, we will provide evidence of how growth factors secreted by glial cells, such as epidermal growth factor and transforming growth factors, control cerebellar organogenesis. Finally, we will argue that glia are a key mediator of cerebellar development and that identification of molecules and pathways involved in neuron-glia interactions may contribute to a better understanding of cerebellar development and associated disorders.

Keywords Cerebellar development . Thyroid hormones . Epidermal growth factor . Transforming growth factor beta 1 . Migration . Synapse

Introduction ataxias, developmental disorders, autism [6, 7] attention defi- cits, hyperactivity, schizophrenia, as well as neuroendocrine A relatively simple structure with well-defined anatomy and disorders and pediatric tumors [8]. physiology, the cerebellum has long been recognized as the The development of the cerebellum is a rigidly regulated primary center of motor coordination in the central nervous process giving rise to a characteristically organized laminar system (CNS). Classically, the cerebellum acts as a motor structure. This structure includes the formation of a cerebellar coordination center, using sensory stimuli from the periphery territory along the antero-posterior and dorsal-ventral axes of of the body to refine movement and balance [1]. However, it is the neural tube, initial specification of cerebellar cell types, now known that the cerebellum is also related to various brain their subsequent proliferation, differentiation and migration, mechanisms, such as cognitive and emotional responses and, finally, the formation of the cerebellar circuit [9]. [2–5]. Cerebellar glial cells have been shown to play a significant Critical biological processes such as proliferation, differen- role in controlling many of these events as neurogenesis, cel- tiation, migration, and synaptogenesis occur during the devel- lular migration, axonal growth, synapse formation and func- opment of the cerebellum, and dysfunction related to these tion, maintenance of the blood-brain barrier, and control of events can trigger serious neurological problems, such as homeostasis and vascular tone [10–12]. Together with the role of glial cells in brain physiology, emerging evidence from recent decades has associated deficits in glial function with * Flávia Carvalho Alcantara Gomes several neurological diseases that affect specific brain regions, [email protected] such as cerebral cortex, midbrain, and spinal cord [13–16]. 1 Instituto de Ciências Biomédicas, Universidade Federal do Rio de The apparent simplicity and geometric disposition of the Janeiro, Rio de Janeiro, RJ, Brazil cerebellar cortex have appealed to many scientists as an 1018 Cerebellum (2019) 18:1017–1035 excellent model for the study of mechanisms involved in the Here in this review, we will highlight three other molecules, development of the CNS [17]. The cerebellum is divided mac- essential for cerebellar morphogenesis: thyroid hormone roscopically into three regions, a medial portion called the (TH), epidermal growth factor (EGF), and astrocytic vermis, lateral regions on either side of the vermis in areas transforming growth factor beta-1 (TGF-β1). called the paravermis, and lateral portions adjacent to each TH deficiency during the perinatal period dramatically af- paravermis, known as the hemispheres [18]. The conserved fects cerebellar morphogenesis [47, 48], resulting in various foliation patterns in the mammalian adult cerebellum are ana- abnormalities, including neuronal cell loss, impaired neurite tomically divided into ten lobes [9, 19, 20], which are sepa- growth and myelination, delayed proliferation and migration rated by a series of fissures that extend to a specific depth in of granule cells, reduced number of synapses between granule the cerebellum, giving each lobe a unique shape [20, 21]. The cells and PCs, aberrant patterns of connections, and loss of lobes are divided into sublobes that vary in number according correct cerebellar structural organization patterns [48–52]. to the species [19, 22](Fig.1a, b). Furthermore, hypothyroidism is associated with abnormalities Microscopically, the cerebellar cortex of adult mammals is in the cerebellar cortex, such as persistence of the EGL and composed of eight different neuronal populations: Purkinje altered foliation pattern [52–57]. The majority of cell subsets cells (PCs), Golgi cells, granule cells, basket cells, stellate in the developing cerebellum express the receptors for this cells, Lugaro cells, unipolar brush cells, and candelabrum cells hormone, thyroid receptors TRs [58]. Activation of these re- [17, 23, 24] and astroglial and oligodendroglial cells that are ceptors leads to activation of different signaling pathways re- classified into four main distinct categories according to their lated to proliferation and differentiation processes [59, 60], morphology: fibrous astrocytes and oligodendrocytes, proto- such EGF pathway, as it will be discussed later in this review. plasmic astrocytes (velate), neuroepithelial cells known as Transforming growth factor betas (TGF-βs) are multi- Bergmann’s glia (BG), a specialized glial type, and a fourth functional growth factors that participate in the regulation type of cerebellar glia neglected by scientists, the cells of of key events related to development, disease and tissue Fañanas [25], classified as a subtype of BG [26–28](Fig.1c). repair. In the brain, TGF-β1 has been widely recognized Together with the various neuronal types, the astroglial and as an injury-related cytokine, specifically associated with oligodendroglial cells give the cerebellum its anatomical and astrocyte scar formation in response to brain injury. In the functional complexity. The cerebellar cortex is composed of a last decade, however, evidence has indicated that in addition very basic structure formed by three distinct layers, each com- to its role in brain injury, TGF-β1 may be a crucial regulator posed of distinct types of cells. The most superficial layer, the of cell survival and differentiation, brain homeostasis, angio- molecular layer (ML), is a region of low cell density but a high genesis, memory formation, and neuronal plasticity [61]. incidence of synapses and is formed by granule cell axons, the The expression profile of the Smad family varies greatly dendritic processes of PCs, basket cells, and stellate cells. The between different CNS regions of the adult mouse. In the cell bodies of PCs are organized into a single row. These cells cerebellum, Smad2 and Smad3 are highly expressed in the together with candelabrum cells and aligned with the BG cre- ML and the PCL and less abundant in the GL, while an ate a monolayer between the ML and the granular layer (GL) inverse expression pattern is observed for Smad4, whose known as the Purkinje cell layer (PCL). Above and below this expression is lower in the ML and the PCL and increased layer is found a second type of glia, the Fañana cells [25, 29]. in the GL [62]. Additional evidence of the role of TGF-β in The GL is the deepest layer, with the highest cell density, cerebellar development comes from genetic/molecular containing millions of granule cells, Golgi cells, Lugaro cells, models. Deletion of Smad4 causes irreversible cerebellar velate astrocytes, and a unipolar brush cell [30–33]. Below the damage, such as a decrease in the number of PCs [63], three layers is the white matter containing a dense network of whereas disruption of Smad2 leads to aberrant development fiber tracts and cells including fibrous astrocytes and oligo- of the cerebellum and early ataxia in mice [64]. dendrocytes [34]. There is evidence that glial cells are the main source of The development of the cerebellum is controlled by growth factors/trophic factors and extracellular matrix mole- many signaling molecules: fibroblast growth factor cules in the CNS. Through the production of active molecules, (FGF) family participates in the scaffold formation of glial cells control several steps of brain development, includ- BG fibers and in the migration of granule cells, regulates ing cellular migration [12], homeostasis [65], maintenance of the generation and correct positioning of BG among other the blood-brain barrier [65, 66], regulation of neurotransmis- functions [35, 36]; Wnt/β-catenin pathway is critical for sion [67, 68], synapse formation [11, 69], and elimination cerebellar foliation and lamination [37]; Notch signaling [70], among others. However, less evidence is available about plays a key role in the development of BG phenotype and the role of neuron-astrocyte interactions during development regulation of granule cell migration [38–42]; and Sonic and their impact on diseases of the cerebellum. hedgehog (Shh) modulates the proliferation of GCPs and In this review, we will focus on the mechanisms un- determines the pattern of cerebellar foliation [21, 43–46]. derlying neuron-astrocyte interactions during cerebellar Cerebellum (2019) 18:1017–1035 1019 a b R

VIII A P VII V C

VIa IX VI

H VIb V H X IV/V VII R VIII III L M IX I/II C c

ML

PCL

GL

WM

Fig. 1 Cerebellar architecture and development. a Dorsal view of an caudal; H, hemisphere; L, lateral; M, medium; P, posterior; R, rostral; adult mouse cerebellum. b Nissl coloring of sagittal section of the P6 V, vermis; 1, Fañana cell; 2, Purkinje cell; 3, stellate cell; 4, Bergmann mouse cerebellum showing the structure of the lobules (× 4 glia; 5, basket cell; 6, candelabrum cell; 7, Golgi cell; 8, Lugaro cell; 9, magnification). The lobules are labeled with Roman numerals. c granule cell; 10, unipolar brush cell; 11, protoplasmic astrocyte; 12, Scheme representing the structure of the cerebellar cortex with the oligodendrocyte; 13, fibrous astrocyte; ML, molecular layer; PCL, arrangement of the cells in their layers. Abbreviations: A, antero; C, Purkinje cell layer; GL, granular layer; WM, white matter development and function. More specifically, we will Cerebellar Glia: Astrocytes and Bergmann Glia provide data on how astrocytes, THs, and growth fac- tors secreted by glial cells, such as EGF and TGF-β1, The majority of cerebellar astrocytes are generated during control cerebellar organogenesis. Finally, we will present embryonic development and in the late postnatal period [71, and discuss evidence of how deficits in neuron-glia in- 72]. Chronologically, the astrocytes appear after the major teractions impact cerebellar function and may contribute periods of neurogenesis and neuronal migration are completed to cerebellar dysfunction. in the mammalian brain. Astrocyte differentiation and 1020 Cerebellum (2019) 18:1017–1035 maturation are distinct between the different regions of the several additional studies that show that abnormal glial brain, and these events are essential for several processes of development leads to defective cerebellar formation [82, CNS ontogenesis [73]. 83]. Furthermore, it has been demonstrated that astro- There is evidence that during development of the cerebel- cytes give rise to GCPs in the EGL [74], providing lum, the progenitors in the EGL generate granule cells and further evidence that astroglial cells are pivotal to the glial fibrillary acidic protein (GFAP)-positive cells [74], but generation of neurons, as has been observed in other it is unclear whether these cells that are multipotent in vitro structures of the CNS. In this section, we will review [75] can differentiate into mature astrocytes in vivo. several studies that support the hypothesis that astroglial Altman and Bayer identified two main sites of cellular cells are major regulators of cerebellar development. proliferation in the embryonic ventricular neuroepithelium: As mention earlier, the cerebellar cortex has two a posterior, located anterior to the rhombic lip, and an main astroglial types: BG, a specialized type of astro- anterior one, near the isthmus and the superior cerebellar cyte located at the PCL, and velate astrocyte, a proto- peduncle. They also observed that the cells continue di- plasmic astrocyte present at GL [84, 85] typical of areas viding into the overlying tissue and therefore have sug- of high neuronal density such as the cerebellar cortex. gested that cerebellar glia can be generated by progenitors In contrast with fibrous astrocytes that are found in that proliferate in the cerebellar parenchyma [76]. white matter and are much less elaborate than protoplas- Studies of Notch activity in the cerebellar primordium in- mic astrocytes, processes from velate astrocytes are lon- dicated that both cerebellar neurons and glia can originate ger, are of higher caliber and establish contacts with the from common ancestors [77]. Notch is expressed from nodes of Ranvier of neuronal axons. Velate astrocytes E10.5 in mouse ventricular neuroepithelium, but not in the have large cell bodies, and their many thick extensions rhombic lip. The lack of Notch results in early neuronal dif- create a shrub-like structure [84, 86]. The large mem- ferentiation [77, 78], leading to a rapid decrease of progenitor brane extensions wrap granule cells synapses with cells and a hypomorphic cerebellum [78]. In E9.5, the consti- mossy fibers, separating the glomeruli into distinct enti- tutive activation of Notch in the cerebellar progenitors favors ties [87]. The astrocytes enclose the glomeruli and con- the generation of astrocytes at the expense of the neurons [77]. trol the infiltration of dendrites and axons in this struc- Classical works propose that BG, a specialized type ture [88] and also limit the diffusion of neurotransmit- of astrocyte, derive directly from the radial glia through ters away from the synaptic location, isolating synaptic the retraction of ependymal processes, the progressive complexes that convey different information [89, 90]. displacement of the cellular body toward the cortex Velate astrocytes also assemble around blood vessels, and the maintenance of basal processes adjacent to the forming flattened sheet-like board around the basal lam- pial surface [79–81]. The ontogenesis of the cerebellum ina [90, 91] making part of the blood-brain barrier. depends greatly on the interactions between neurons and Recently, it was observed that each velate astrocyte glial cells, as we shall see below. forms a syncytial network through gap junctions with Glial cells were previously regarded as merely support- nine neighbors, resulting in a highly coordinated struc- ive and passive elements of the CNS. The past decade, ture in the cerebellum [92]. In fact, this astrocyte syn- however, has been marked by a thorough revisitation of cytial organization may be responsible for controlling the role of these cells, not only in healthy brains but also the diffusion of small molecules throughout neighbor in brain disease. Astroglial cells have emerged as key cells, such as a calcium waves in the cerebellum [93], mediators of brain development, function, and plasticity, which may be associated with diverse cerebellar astro- highlighting the critical need to better characterize the cytic functions, as blood flow and synaptic modulation. mechanisms underlying their development and interaction Additionally, as will be described later in this review, with neurons. While there is compelling evidence regard- our group has shown that cerebellar astrocytes are asso- ing the impact of astroglia function in the cerebral cortex ciated with neuronal proliferation [94], differentiation and hippocampus [11, 12, 65, 66], there is still a lack of [95, 96], GCP migration, BG development [97], and data on the role of astroglial cells in the cerebellum and cerebellar synaptogenesis [98]. However, more informa- cerebellar dysfunction. tion about cerebellar astrocytes is scarce, and there are In cerebellar development, glial cells are of the ut- still more questions than answers about their role in most importance, as evidenced by conditional ablation cerebellar physiology. of GFAP expressing cells in the cerebellum [82]. The BG are a unique glial subtype of the cerebellar cortex ablation of 50% of GFAP-positive cells shortly after that are generated from the radial glia (RG) of the ven- birth leads to a hypoplastic cerebellum without well- tricular zone of the medial portion of the fourth ventri- defined histological layers, with decreased granule cell cle [99] This cell type presents its cell body in the PCL numbers and ectopy of PCs. This is supported by and emits its processes to the pia matter surface, Cerebellum (2019) 18:1017–1035 1021 passing through the ML, constituting the glial limiting leads to a reduction in GCP proliferation and an in- membrane [100]. The BG role is similar to RG support crease in GPC differentiation status, resulting in de- in cortical development. Both types of glial cells have creased EGL thickness. This demonstrates that BG cell radial morphology and act as a framework for neuronal signaling maintains CGP proliferative capacity [16]. migration, guiding neurons in their arrival at their cor- In the PCL, BG provide structural support for PC dendritic rect locations. However, BG and RG have a fundamen- extension [103] and act as the third element of the PC synap- tal difference; after the migratory period, RG in the ses, supporting synapses made between PC and their patterns cerebral cortex disappear by differentiating into astro- [80] and wrapping Purkinje dendrites and synapses from the cytes [101–103]. Unlike RG, BG do not disappear in early postnatal period onward [121]. These processes are re- the adult and play a key role in cerebellar function in sponsible for maintaining the rapid clearance of synaptic glu- adulthood [79]. Interestingly, BG continue to display a tamate. If any alteration occurs in this structure, BG glutamate guiding capacity for neurons as showed by Sotelo and uptake capacity is reduced, leading to the formation of aber- coworks, that demonstrated that grafted PC in the adult rant synapses between PCs and climbing fibers [108, 121, cerebellum uses BG for their migration and integration 122] which can be associated to comportamental alterations. in the cerebellar cortical circuitry [104, 105]. Accordingly, it has recently been shown that a mouse model BG play an important role in almost all phases of for leukoencephalopathy exhibited abnormal development of cerebellar corticogenesis, including foliation, orientation, BG, which led to ectopic localization of BG cell bodies and stratification of granule cells, neuronal migration, deficits in synaptic wiring. In these mice, PC dendrites are not neurite outgrowth, and synaptogenesis [106]. BG are completely wrapped by BG cell processes and atypical syn- also important in adult cerebellar function, including aptic formation is observed, most likely due to the inability to extracellular ion homeostasis [107], synaptic stability eliminate unwanted synapses [123]. Besides glutamatergic [108, 109], plasticity [110, 111], metabolic function, connections, BG also participates in GABAergic innervation and neuroprotection [112, 113]. formation between PC and stellate cells. Ango and coworkers During cerebellar development GCPs are found in have shown that BG processes serve as a scaffold for stellate close contact with glial cells [106, 114], and BG play axons to reach PC dendrites and properly form GABAergic a major role by providing a scaffold for GCPs migration connections, leading to a precise pattern of circuit organiza- from the EGL to the IGL, which is essential for GCPs tion [124]. survival [115, 116] . In 2014, Li and coworkers dem- Moreover, it was suggested that BG had a role in gen- onstrated that mice lacking Shp2 protein present a erating other cell types in the mature cerebellum. In the smaller cerebellum without visible foliation. These ani- mature structure, BG express molecules such as Sox1 and mals have impaired BG formation, and as a result, al- Sox2, which are involved in cell self-renewal and mainte- though GCPs proliferate at the EGL, they do not mi- nance of undifferentiated neural progenitors [125, 126]. grate to the IGL, leading to failure in folia formation Another finding that corroborates the hypothesis of BG as [117]. Similarly, another group has shown that when BG cerebellar progenitor was that the cerebellum of adult rab- cells have disrupted fibers, which fail to reach the sur- bits is apparently able to give rise to synantocytes [127] face of posterior lobes, ectopic granule cells are detect- which are newly described NG2-positive glial cells that ed, and PCs are irregularly aligned [118]. Additionally, have the ability to proliferate. In vitro, they differentiate our group and others have shown that THs strongly into astrocytes and oligodendrocytes, but in vivo, they only influence BG formation and, in turn, cerebellar morpho- express markers of cells involved in the oligodendrocytic genesis. The expression of a non-binding TRβ receptor, lineage, such as O4 and Gal-C [128, 129]andthetheir which impairs 3,5,30-triiodothyronine (T3) activity, capacity to proliferate is not clear. Grimaldi and Rossi have leads to BG defects and, consequently, malformations shown that infusion of growth factors in physiological con- in cerebellar laminar organization [119]. Moreover, the text or PC depletion did not induce a significant prolifera- expression of a truncated thyroid receptor α1(TRα1) tion even though there are mitotic cells in the cerebellum, isoform in BG results in BG mislocalization, and the such as NG2-positive cells [130]. Nevertheless, other appearance of twisted fibers that do not reach the pial groups have shown that, instead of BG cells, there are surface impairs GCP migration from the EGL to the progenitor cells in the cerebellum that give rise to astroglial IGL and thus the maturation of granule cells in the cells of the adult. Parmigiani and collaborators have shown IGL [120]. BG also participate in cell proliferation and that the postnatal cerebellum presents two pools of differentiation in the cerebellum. Cheng and coworkers astroglial-like progenitors cells. One gives rise to have recently demonstrated that Shh signaling in BG GABAergic interneurons and astrocytes at the prospective controls GCP proliferation. Inhibition of Shh signaling white matter, while the other astroglial-like progenitors gen- by ablation of Shh activator, Smoothenerd (Smo) in BG erate astrocytes and BG in the PCL [131, 132]. Also, after 1022 Cerebellum (2019) 18:1017–1035 depletion of granule cells in the EGL of neonate mice, formation, the initial structure of the cerebellum is replaced by immature astrocytes suffer an adaptive reprogramming and a foliated pattern that is conserved through evolution [139] change their progenitor cell markers. This change leads to (Fig. 2). expression of GCP genes by these newly differentiated cells and repopulation of the EGL [133]. Furthermore, Ahlfeld and colleagues demonstrated that there are Sox2- Thyroid Hormones and Epidermal Growth Factor positive cells intermingled with BG, which are responsible for neurogenesis induced by exercise in the adult cerebel- In rodents, after birth, GCPs extensively proliferate in the EGL lum [134]. In that way, it seems that the cells that are during the first postnatal week [43, 140–142]. During the second responsible for the generation of newly cells in the adult postnatal week, GCPs reach peak proliferation, differentiating would be the progenitors cells present in the adult during migration through the ML and PCL to reach their mature cerebellum. state in the IGL by the end of the third postnatal week [9]. This In the cerebellum, beyond the astrocytes and the BG, there period coincides with the accumulation and differentiation of is another type of radial astrocytic cell, the Bfeathered cell^ of precursors, radial neuronal migration, cerebellar foliation, and Fañanas [25–27, 135], a subtype of short BG cells [28]often the maturation of PCs [143–145]. These steps are tightly regu- overlooked by scientists. These cells are very similar, but not lated by a balance between intrinsic and extrinsic molecules, identical to BG cells, which makes their identification difficult growth factors and hormones that trigger multiple signaling path- by simple morphology. A recent work reveals two potassium ways [21, 43, 46, 48, 95, 97, 146–150]. In the cerebellum, glial channel-related polypeptides, Kv2.2 and calsenilin (KChIP3) cells are reported to produce several molecules that are associated as potential marker proteins for these cells. Through immuno- with these developmental events, such as components of the cytochemistry assays, it was established three morphological extracellular matrix: laminin, fibronectin, and proteoglycans criteria that may differentiate the BG from the cells of [95, 151–155]; growth factors: EGF [95, 97], FGF [154], Fañanas. They differ in size and shape from cell bodies: BG TGF-β1[98], and Shh [119]. In this section, we review how exhibit an epithelioid appearance whereas Fañanas cells are cerebellar glia control GPCs proliferation, differentiation, and smaller and resemble fibrocytes. Fañanas cells are located in a migration through the activation of two of these molecular sig- much wider range above and below PC’s and do not form a naling pathways: THs and EGF. single row as the BG; further, BG send unbranched processes THs, thyroxine (T4) and T3, are key modulators of cellular to the pial surface while the Fañanas cells do not [29]. metabolism and are essential for embryonic development. T4 is able to cross the blood-brain barrier [156] and then is taken up by astrocytes. Once inside astrocytes that express type 2 Signaling Pathways Involved iodothyronine deiodinase, T4 is deiodinated to produce T3 in the Development of the Cerebellum: Role [157] which is considered a biologically active hormone. of Astrocytes THs exert their functions through the nuclear TH receptor (TR: TRα1, TRβ1, and TRβ2), a ligand-dependent transcrip- Although the cerebellum is one of the first structures of the tion factor [158]. THs control several steps of cerebellar de- CNS to differentiate itself, it reaches its mature configuration velopment and function [119, 159, 160], including glial cell only a few months after birth in humans and 15 days after birth differentiation and function; neuronal migration, axonal arbor- (P15) in mice [136]. This period of postnatal development has ization, and growth; and synapse formation [95, 097, 98, 16 ]. advantages and disadvantages since the cerebellum is an eas- A lack of TH signaling during fetal and early postnatal periods ily accessible structure, it is widely used in experimental stud- results in severe mental retardation, such as cretinism in ies, but its developmental profile leaves the cerebellum vul- humans [47, 159, 161, 162]. nerable to irregularities throughout development. Several evidences from transgenic animals studies, knockout The beginning of rodent cerebellar development occurs or knockin for TRs, strongly support a role of TH in the devel- around the eighth embryonic day (E8) and ends at P15 when opment and function of the cerebellum [119, 120, 163, 164]. For the cerebellum exhibits an organization with all its cell types example, mutation of TRα1 in PCs leads to severe deficits in PC distributed in three layers (ML, PCL, GL) and a morphology morphogenesis [120] and expression of mutant TRβ1 as early as that correlates with the location of specific cerebellar circuits. P2 results in decreased PC dendritic arborization and reduced The massive postnatal proliferation of GCPs in the EGL granule cell migration [164] leading to cerebellar ataxia. We also and their subsequent migration lead to extensive growth of the reported that target inactivation of TRβ-TH binding leads to a cerebellum after birth [137], and the disappearance of the EGL smaller cerebellum area characterized by impaired lamination at the end of the third postnatal week and the formation of and foliation. Furthermore, TRβ mutant mice presented severe internal granular layer (IGL) [138] gives rise to the stereo- deficits in proliferation of GCPS, arborization of PCs, and orga- typed three-layer structure of the adult cerebellum. After its nization of BG fibers [119]. Cerebellum (2019) 18:1017–1035 1023

P6 P12 P15 AD

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EGL EGL ML ML

ML ML PCL PCL PCL PCL

IGL IGL IGL GL

EGL

ML

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Granule Cell Precursor Migratory Granule Cell Bergmann Glia Cell Purkinje Cell Granule Cell

Fig. 2 Sagittal section of the mouse cerebellum and scheme representing Bergmann cells as support. Abbreviations: AD, adult; EGL, external the sections (× 20 magnification). The granule cells proliferate in the EGL granular layer; GL, granular layer; IGL, internal granular layer; ML, and migrate through the Purkinje cells layer to form the IGL, by using the molecular layer; P, postnatal day; PCL, Purkinje cell layer

Most of the effects of THs on cerebellar development and the hippocampus [179, 180], neurite outgrowth of forebrain neu- function have been attributed to their direct actions on neurons rons [181], migration and proliferation of stem cells in adults [47, 164–166]. Alternatively, we and other authors have shown [182–184], and long-term potentiation in the hippocampus that TH actions on cerebellar development can be mediated by [185]. Furthermore, EGF also plays a key role in cerebellar and growth factors and extracellular matrix molecules produced by hippocampal astrocyte proliferation [186, 187] and differentia- astrocytes. For example, T3 induces EGF, tumor necrosis factor tion in the telencephalon and hippocampus [187 , 188]. Further beta (TNF-β), and FGF release from astrocytes [94, 96, 154, support for the action of EGF in CNS comes from EGFR dele- 167]. Additionally, this hormone has been demonstrated to reg- tion which leads to embryonic defective cortical neuronal migra- ulate the cerebellar expression of extracellular matrix and adhe- tion [189], early postnatal neurodegeneration and impaired astro- sion molecules that are important for neuronal migration and cyte proliferation [190]. development, such as tenascin-C [156], reelin [168], laminin, Our group has provided growing evidence over the last and fibronectin [95, 154, 169, 170]. Our lab demonstrated that decades strongly indicating a role for EGF produced by glial conditioned medium derived from T3-treated (T3CM) cerebellar cells in cerebellar development. By using different methodo- astrocytes induced cerebellar precursor proliferation and morpho- logical approaches, we have found that astrocytes stimulated logical differentiation in vitro [94]. Our work was of the first to with T3 release EGF and increase GCP proliferation through demonstrate an indirect effect of T3 mediated by cerebellar as- PKA pathway activation. Additionally, EGF is also able to trocytes instead of an exclusive and direct role for the hormone in increase extracellular levels of laminin and fibronectin in the neuronal function. This indirect effect was mainly mediated by cerebellum, two extracellular matrix molecules that provide enhanced production of EGF by astrocytes in response to T3 directional cues for neurite development [169, 170], leading [94–96]. to GCP neurite outgrowth by stimulation of MAPK and PI3K EGF is a growth factor that was initially noted for its ability to signaling [95, 96]. In agreement with these data, Mendes-de- stimulate ectodermal and mesodermal cell proliferation [80, Aguiar and coworkers observed that astrocytes from hypothy- 171–173] by stimulating EGF receptors (EGFR). Once activated, roid rats present reduced fibronectin and laminin distributions, these receptors stimulate a series of signaling pathways, such as leading to impaired neuritogenesis of cerebellar neurons ERK MAPK, AKT-PI3K, and PLC-1-PKC [174], that can lead [191]. Although it is well known that hypothyroidism results to global phosphorylation of approximately 2000 proteins within in abnormal PC dendritic arborization [47, 164, 166, 192], our the cell [175]. Currently, EGF has mainly been observed to be data shed light on astrocytes as important targets of this phe- related to a series of human cancers, and the development of nomenon. Furthermore, astrocytic EGF is also a key molecule cancer therapies based on inhibition of these receptors has in- in BG differentiation and neuronal migration. Incubation of spired extensive research [176, 177]. In the CNS, EGF induces cerebellar explants from P7 rats with conditioned medium neuronal differentiation in the neural tube [178], proliferation in derived from T3-treated astrocytes induced elongation of 1024 Cerebellum (2019) 18:1017–1035

BG processes and increased neuronal migration by activation We previously demonstrated the involvement of TGF-β1 of MAPK in an EGF-dependent manner [97]. This is support- pathways in several events of brain development and disease, ed by the location of EGFR mRNA within the EGL at the including gliogenesis and RG differentiation [94, 160, 209, inner premigratory area. Interestingly, EGFR mRNA levels 210]; astrocyte differentiation [211, 212]; angiogenesis decrease when migratory cells arrive in the IGL as a result [213]; synapse formation [11, 69, 98, 208, 214]; and in of granule cells migration [193, 194]. Furthermore, EGFR Alzheimer’s disease (AD) [13], and septic encephalopathy activation is related to neural progenitors and astrocytes mi- [215]. gration in the telencephalon [195]andcortex[196, 197]. Recently, our group identified TGF-β1 as a new mol- Moreover, EGFR KO mice have ectopic neurons in the white ecule involved in the regulation of excitatory and inhib- matter of the hippocampus, suggesting that EGF plays a role itory synapse formation in the cerebral cortex and hip- in cellular migration [190]. pocampus. We have shown that TGF-β1 induces the Together, our data propose a mechanism by which THs formation of excitatory functional synapses in mice influence cerebellar development: first, astrocytes uptake T4 through induction of the secretion of the NMDA recep- hormone and metabolize it to T3, which is released and acts in tor co-agonist, D-serine [69 ]. In contrast, induction of a paracrine way on astrocytes and on neurons. By activating inhibitory synapse formation by TGF-β1 is dependent different downstream pathways, EGF influences neuronal on glutamatergic activity and activation of CaM kinase proliferation and differentiation (PKA pathway), neurite II, which leads to the localization and cluster formation growth (through modulation of extracellular matrix molecules of the synaptic adhesion protein, Neuroligin 2, in inhib- via MAPK and PI3K pathways), granule cell migration, and itory postsynaptic terminals [69]. BG process elongation (Fig. 3). However, the mechanisms by More recently, we have shown that TGF-β1secretedby which these signaling pathways modulate these processes are astrocytes plays a key role in cognitive processes under phys- still an open question, and a demonstration of this proposed iological and pathological conditions. We have demonstrated model in in vivo models are yet to come. that TGF-β1 secreted by astrocytes improves the memory of healthy adult mice [208] and protects against memory loss in Astrocytic Transforming Growth Factor Beta-1 AD animal model [13]. TGF-β1 and Smads are expressed in different regions of The correct development of the CNS depends on the requisite the CNS during development and in adulthood [216–218]. In number and function of synapses that are critical for the for- the adult rat cerebellum, the mRNA for TGF-β1isfoundin mation of neural circuits. However, due to the large number of the molecular layer, in the PCL, and in the GL [218]. We have synapses and circuitry types in the mammalian brain, much shown that TGF-β1 is expressed during the development of remains to be discovered about the mechanisms that control the cerebellum by granule cells and astrocytes [98, 214]. the development of these events. Granule cells and PCs express the TGF-β1receptor(TβRII) Synaptic connections are formed, maintained or eliminated by in vivo, suggesting that these cell types are targets of this molecules called synaptic organizers that collectively define the factor [98](Fig.4). function of neuronal circuits. In the cerebellum, these organizers The TGF-β1 pathway is triggered by its binding to TβRII, include several secreted factors and adhesion molecules such as which recruits TβRI, leading to its self-phosphorylation and neurexins and neuroligins that determine the properties of spe- the initiation of a phosphorylation cascade that involves cific synapses [198, 199]. Such organizers include brain-derived downstream effector proteins from the Smad family. After neurotrophic factor (BDNF) that is secreted by PCs and promotes Smad2/3 recruitment and phosphorylation, these proteins the elimination of climbing fiber synapse [149] glutamatergic form a complex with Smad4 that translocates to the nucleus receptors of the NMDA type that are essential for cerebellar and binds to TGF-β1-activated genes [61, 219]. synaptic elimination [200] and functional inhibition of These studies reinforce the importance of the role of GABAergic synapses [201], and cerebellin 1, which is specifi- TGF-β1 in cerebellar development; however, less is known cally required for the formation and maintenance of parallel fi- about its role in astrocyte involvement in cerebellar synapse bers synapses, together with the δ2 glutamate receptor (GluD2) formation. [202, 203], among others. Genomic studies from our group and others has revealed Astrocytes regulate the maintenance and elimination of syn- that astrocytes from the cerebral cortex, hippocampus, mid- apses by regulating the overall architecture and activity of neural brain, and cerebellum vary considerably in their gene expres- circuits through direct contact [204–207]. Thus far, there is a lack sion profile for some synaptogenic factors, including TGF-β1, of information about astrocytic-derived factors that control cere- SPARC, Hevin, glypicans 4 and 6, and TSP. These data sug- bellar synaptogenesis, although we recently identified TGF-β1 gest that astrocytes from these regions have different as an astrocyte-secreted protein that induces synapse formation in synaptogenic potentials [214, 220, 221]. Interestingly, we the cerebral cortex and hippocampus [11, 69, 208]. have observed that cerebellar astrocytes are the most efficient Cerebellum (2019) 18:1017–1035 1025 T3 Signaling Activation

T3

EGF Increase of EGF release by astrocytes

Neuronal differentiation (Martinez & Gomes, 2002;2005) Bergmann Glia elongation Laminin Granule Cell migration (Portela et al., 2010 Matriz GCP Proliferation Martinez et al., 20110) Reorganization (Gomes et al., 1999)

PKA Fibronectin activation

Granule Cell Precursor (GCP) MAPK activation MAPK PI3K Migratory activation Granule Cell

Granule Cell Bergmann Glia

Fig. 3 Thyroid hormone actions in astrocyte functions. T3 hormone acts in a signaling, astrocytes also induce cellular migration and elongation of BG paracrine signaling resulting in increase of EGF secretion from cerebellar processes in a MAPK-dependent manner. Images from https://smart.servier. astrocytes. Once released, EGF induces cellular proliferation through PKA com/. Abbreviation: EGF, Epidermal Growth Factor; GCP, granule cell activation and extracellular matrix reorganization which results in cellular precursor; T3, Thyroid hormone differentiation by MAPK and PI3K signaling. Through activation of EGF at inducing excitatory synapse formation when compared with The effect of TGF-β1 on synapse formation is context and astrocytes from other regions [214]. cell dependent. TGF-β1 is unable to induce the formation of 1026 Cerebellum (2019) 18:1017–1035

a Calbindin b TβRII c Calbindin TβRII

d βTubIII e TβRII f β Tub III TβRII

Fig. 4 TGF-β receptor’s distribution in the cerebellum, in vivo and PCL. Cultured granule cells obtained from P6 mice present TβRII distri- in vitro. a Sagittal sections of the adult mouse cerebellum stained with bution throughout the neuronal membrane. The images show confocal anti-calbindin and b anti-TβRII. c TβRII colocalizes with calbindin in the microscopy analysis (a–c) excitatory synapses between neurons of the retinal ganglion thus affecting membrane excitability, mitochondrial fu- [222]; however, it induces synapses between neurons from the sion, and intracellular homeostasis [225]. hippocampus [223], cerebral cortex [11, 69], and the neuromus- These findings, together with the fact that TGF-β1 and its cular junction [224]. However, the mechanisms underlying these receptors are expressed during the synaptogenic period of cer- events may vary between regions. We have also shown that ebellar development in vivo and that granular neurons are TGF-β1 promotes the formation of excitatory synapses between responsive to TGF-β1 signaling, support the concept of GCPs [98]. GCPs express the TβRII, and exposure of GCP TGF-β1 as a key molecule in the formation/function of syn- cultures to TGF-β1 increases the level of p-SMAD, suggesting apses in the cerebellum. that these cells are responsive to TGF-β1. Loss of endogenous TGF-β function in culture (by pharmacological blockade) de- creases the number of synapses, while treatment of neuronal Glia and Diseases: Impact on Cerebellar cultures with TGF-β1 increases pre- and postsynaptic protein Dysfunction levels, suggesting that the TGF-β pathway is important to main- tain cerebellar synapses. Accumulating evidence has shown that astrocytes are important Additional evidence regarding the role of TGF-β1in components of numerous pathologies of the CNS [226, 227], cerebellar synapse formation and function has come such as Down’s syndrome [228, 229], Parkinson’s disease from the demonstration that TGF-β1actsontheelec- [230], epilepsy [231, 232], Huntington’s disease [233, 234], trophysiological properties and maturation of GCPs and AD [13, 235]. Because glial cells play several major roles from rats by increasing the expression of the GABA in cerebellar development and function, we hypothesize that as- receptor α-6 subunit and reducing the influx of extra- trocytic dysfunctions may play a key role in the development of cellular calcium [146]. Furthermore, TGF-β1 decreases cerebellar diseases. Although this issue has been explored in the gene and protein expression of the major intracellu- relation to several CNS regions, glial alterations in cerebellar lar calcium transporters in rat cerebellar granule cells, pathologies have not received sufficient attention. Nevertheless, Cerebellum (2019) 18:1017–1035 1027 some groups have implicated glial cells in diseases of the cere- Astrocytes from different brain regions present distinguish- bellum, such as ataxia, leukoencephalopathy, autism, and atten- able features with respect to metabolism, developmental ori- tion-deficit/hyperactivity disorder (ADHD) [236–239]. In agree- gin, neurogenic and proliferative potential, protein expression ment with our hypothesis, it was recently shown that cerebellar profile, physiological properties and synaptogenic potential astrocytes from GIT1 KO mice, an animal model for ADHD, [198, 246–251]. They also present different responses to path- have decreased intracellular GABA content and impaired tonic ological insults and aging. Boisvert and collaborators have inhibition capacity. Consequently, granule cells have a higher recently shown that gene expression during aging is differen- excitation/inhibition ratio, which could be associated with the tially regulated in astrocytes from the cortex, hypothalamus, hyperactivity observed in ADHD [239]. Regarding autism, and cerebellum. Moreover, the transcriptome status of astro- Wegiel and coworkers have reported that autistic patients exhibit cytes from different areas of the brain (cerebellum, cerebral several alterations in BG morphology, such as loss of their ver- cortex, hippocampus and spinal cord) also exhibited a distinct tical processes and impaired cell body arrangement in the ML. pattern of expression in a model of multiple sclerosis [248]. These subjects also show profound disorganization of granule The heterogeneity of astrocytes in response to pathological cells, mainly due to BG alterations, since BG are essential for insults and aging leads to a question of whether astrocyte diver- granule cell migration [240]. Likewise, two different groups have sity might contribute to different vulnerabilities of brain areas to identified that autistic patients exhibit higher levels of GFAP specific diseases. For example, AD does not affect the brain mRNA in the cerebellum [241, 242], which may indicate glial uniformly. During its development, the appearance of Aβ activation in this disease. plaques follows a specific timeline; in initial stages, Aβ plaques Another disease linked to cerebellar alterations and severely are detected in the neocortex and hippocampus, while only in affected by glial dysfunction is hypothyroidism. As discussed in later periods are these plaques observed in the cerebellum of AD the previous sections, this disease leads to several deficits in patients [252]. In APP/PS1 mice, an animal model for AD, the cerebellar development and is associated with ataxia and cretin- onset of deposition of Aβ plaques in the cortex and hippocampus ism [47, 48, 243]. Our group investigated cerebellar glial cells in occurs within 2 months postnatally, and at 8 months, these re- transgenic mice that express a natural human mutation (Δ337 T) gions are replete with deposits of Aβ plaques [253]. However, in in the TRβ locus, resulting in the expression of a non-binding the cerebellum, Aβ deposits are detected only after 12 months, receptor and thereby impaired T3 action. These mice present when these animals begin developing motor deficits. An increase dramatic alterations in cerebellar glia development and morphol- in astrocytic glial reactivity is observed in the cerebellum after ogy, such as poor radialization of BG cells in vivo, a reduction in 18 months [254], whereas in the hippocampus of these animals, the number of BG fibers that reach the pial surface and a de- glial reactivity occurs after 6 months [255]. It is already accepted creased number of BG-derived astrocytes in vitro. These glial that astrocytes play an important role in AD, such as in Aβ alterations led to severe abnormalities in cerebellar foliation, such production and metabolization and the production of inflamma- as fusion of lobules VI and VII at P21 and impaired laminar tory mediators and reactive oxygen species [256–258]. Recently, organization [119]. we have shown that Aβ oligomers (AβO), the main toxin asso- Deficits in glial function are also associated with ataxia. ciated with neuronal deficits in AD [259, 260], impair the Cvetanovic reported that GLAST protein levels are reduced synaptogenic and neuronal protector potentials of hippocampal in the BG of animal models for spinocerebellar ataxia type 1 astrocytes. Our group has also demonstrated that cerebellar as- [236], which can be associated with the loss of PCs in this trocytes have a higher synaptogenic potential when compared model [236, 244], probably due to impaired glutamate uptake with astrocytes from the hippocampus [214]. from BG, which may lead to glutamate excitotoxicity. Taken together, the evidence presented here leads to some Furthermore, astrocytes from A-T mice (a model for ataxia– unanswered questions that need to be addressed in the upcoming telangiectasia studies) present reduced antioxidant capacity years: are cerebellar astrocytes less sensible to AβO insult than due to decreased glutathione levels, which leads to impaired astrocytes from other brain areas? If so, does this make the cer- neuronal support [245]. Moreover, ablation of frataxin, a pro- ebellum more resistant to AD impairments when compared with tein linked to Friedreich’s ataxia, in GFAP-positive cells re- other parts of the brain? We suggest that the answers to these sults in impaired cerebellar development, as shown by the questions will lead to a better understanding of the development absence of GL, disorganized PCL and increased astrocyte of AD. reactivity. Interestingly, glial cells from the forebrain do not exhibit the same sensitivity as cerebellar astrocytes, which show higher oxidative stress levels when compared with fore- Concluding Remarks brain astrocytes [238]. These data suggest that cerebellar as- trocytes might be more susceptible to frataxin impairment The cerebellum presents a diversity of functions, from motor than cortical astrocytes, and this could be a key factor in behaviors involving coordination, learning and balance Friedreich’sataxia. [261–264] to non-motor behaviors such as cognition, 1028 Cerebellum (2019) 18:1017–1035 emotion, language, and spatial navigation [4, 265–267]. This 10. Oberheim NA, Goldman SA, Nedergaard M. Astrocytes. – diversity implies that cerebellar dysfunction is associated with 2012;814:23 45. 11. Diniz LP,Matias ICP,Garcia MN, Gomes FCA. Astrocytic control several diseases, ranging from neurological conditions such as of neural circuit formation: highlights on TGF-beta signaling. ataxia, dystonia, and tremor [268]toneuropsychiatricdisor- Neurochem Int. 2014;78:18–27. ders such as dyslexia, schizophrenia [269], mood disorder and 12. Clarke LE, Barres BA. Emerging roles of astrocytes in neural anxiety [270], attention deficit hyperactivity disorder [271], circuit development. Nat Rev Neurosci. 2013;14:311–21. and autism spectrum disorder [272, 273]. As discussed in this 13. Diniz LP, Tortelli V, Matias I, Morgado J, Bérgamo Araujo AP, Melo HM, et al. Astrocyte transforming growth factor beta 1 pro- review, glial cells participate in numerous events of cerebellar tects synapses against Aβ oligomers in Alzheimer’s disease mod- development and physiology; however, studies regarding glial el. J Neurosci. 2017;37:6797–809. alterations in pathologies related to cerebellar function are still 14. Rakela B, Brehm P, Mandel G. Astrocytic modulation of excitato- scarce. We argue that a better understanding of glial cell phys- ry synaptic signaling in a mouse model of Rett syndrome. Elife. 2018;7.pii: e31629. iology and how these cells are affected in the cerebellum dur- 15. Elgayar SAM, Abdel-Hafez AAM, Gomaa AMS, Elsherif R. ing disease development may provide new insights for im- Vulnerability of glia and vessels of rat substantia nigra in rotenone proved treatment of cerebellar illnesses. Parkinson model. Ultrastruct Pathol. 2018;42:181–92. 16. Wang H, Song G, Chuang H, Chiu C, Abdelmaksoud A, Ye Y, Acknowledgments We would like to thank Dra. Marimélia Porcionatto et al. Portrait of glial scar in neurological diseases. Int J for gently providing us some of the images used in Figs. 1a, b and 2 Immunopathol Pharmacol. 2018;31:2058738418801406. (histological images). This manuscript was edited by American Journal 17. Sotelo C. 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