Introduction

During the last decades, the cerebellum has been studied in great detail, not only to understand its important role in the control of motor coordination, but also because its laminated structure and limited number of cell types have offered a model system to study neuronal development, differentiation, and survival.

Cerebellar granule neurons (CGNs) are the most abundant class of central nervous system neurons. Their progenitors arise prenatally from the rhombic lip, in the boundary of the mesencephalon and metencephalon (mes/met region), to form the external granule layer (EGL). In rodents, the neuroblasts of the EGL proliferate during the first postnatal week. During the second postnatal week, neurons differentiate during their migration through the molecular and Purkinje layers to reach their mature state in the internal granule layer (IGL) by the end of the third postnatal week [1]. During their migration from the EGL towards the IGL, CGNs that fail to receive excitatory inputs from mossy fibers will die by apoptosis [2, 3].

Extracellular Factors Involved in the Proliferation, Differentiation, and Survival of CGNs

The study of the mechanisms and factors involved in CGN proliferation, differentiation, and survival has been facilitated by the possibility to maintain these neurons in culture in the presence of a depolarizing medium. The combined use of CGN primary cultures together with in vivo models has provided a deep knowledge of the main factors sequentially involved in CGN proliferation in germinal layers and their differentiation and survival during migration to the IGL (Fig. 1).

Fig. 1
figure 1

Extracellular factors involved in the proliferation, differentiation, and protection of CGNs at postnatal stages. Main factors introduced in the text involved in the proliferation (prolifer.), differentiation (differ.), and protection (antiapop.) of CGNs at different postnatal stages, from the external granule layer (EGL) to the internal granule layer (IGL). Dashed arrow indicates the migration of CGNs from EGL to IGL. In brackets, the action of glutamate is through the stimulation of NMDA receptor (NMDAR). ML molecular layer, PL Purkinje layer, WM white matter

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Bone morphogenetic proteins (BMPs) seem to be the main factors for the generation of CGN progenitors in the rhombic lip. Work by Hatten and coworkers [4] has shown that BMP6, BMP7, and growth differentiation factor 7 induce the expression of Math1, a transcription factor required for the specification of CGN progenitors in the prenatal cerebellum. Moreover, they demonstrated that BMP-treated explants from the mes/met region were able to generate CGNs when transplanted to the postnatal cerebellum. CGN progenitors proliferate postnatally in the EGL. Several factors have been implicated in their proliferation and in the exit from the proliferative state to start differentiation. Numerous reports have shown that sonic hedgehog (Shh), secreted by Purkinje cells, is the main factor mediating the proliferation of CGN progenitors [5], probably by Nmyc1 activation [6]. However, other factors, such as insulin-like grown factor (IGF) 1 and 2, have also been implicated in the maintenance of the proliferative state of CGN progenitors. Activation of IGF1 receptor by IGF1 and IGF2 enhances Shh-mediated proliferation of purified CGN progenitors [7]. The synergy between Shh and IGFs could be related to the fact that Igf2 expression is modulated by Shh [8]. However, IGF1 and IGF2 are still able to stimulate the proliferation of CGN progenitors (although to a lesser extend) when Shh signaling is impaired [7], indicating that proliferation of CGN progenitors in the EGL does not only depend on the presence of pro-mitogenic factors such as Shh, and that other mechanisms are involved. In fact, it is known that transition from proliferation to differentiation in the EGL starts even in the continuous presence of Shh. Thus, it should be the presence of other factors (anti-mitogens) that drives CGN progenitors towards differentiation. For example, IGF-binding protein 5 is expressed when progenitors start to proliferate (second postnatal week) and it blocks IGF and Shh-mediated proliferation of purified CGN progenitors [7]. Another factor that has been reported to induce cell cycle exit in CGN progenitors is BMP4. The expression levels of BMP4 and Smad1 (an intracellular protein needed for BMP signaling) increase in the EGL during the second postnatal week, and BMP4 promotes neuronal cell differentiation in CGN cultures [9]. Other factors are also known to promote CGN differentiation such as pituitary adenylate cyclase-activating polypeptide (PACAP), vitronectin, Wnt3, or activation of GPR3 receptors, which shows the complexity of the mechanisms involved in this process at the end of the first postnatal week in the EGL [10–13].

Differentiation and maturation of CGNs start during the second postnatal week and occur in parallel to their migration from the EGL towards the IGL [1]. During their migration, stimulation of glutamatergic synapses between mossy fibers, and CGNs are responsible for their survival [2, 3]. It has been reported in vivo that activation of NMDA receptors (NMDAR) is necessary for CGN survival since pharmacological blockade of these receptors produce an increase in the apoptotic rate [14]. Similar requirement has been observed in vitro. Cultured CGNs die from apoptosis when potassium chloride (KCl) concentration changes from 25 mM (K25) to 5 mM (K5). The addition of NMDA rescues CGNs from K5-mediated apoptosis [15, 16]. Moreover, it has been reported that a 24-h exposure to NMDA in immature CGN cultures (2 DIV) is enough to promote a long-lasting neuroprotective effect since cells survive for up to 8 DIV in K5 [17]. Although different signal transduction pathways have been related to the protective effect of NMDA on differentiating CGNs (see below), several studies have shown that NMDA-dependent release of the neurotrophin BDNF is a major factor in CGN survival. Blocking TrkB receptor activation, by either antagonists or BDNF antibodies, produces an important reduction in NMDA-mediated neuroprotection [17–19].

CGN primary cultures have also been used to search other pro-survival factors. Interestingly, some of the factors that are involved in the proliferation of progenitor cells were also related to survival of CGNs during their migration towards the IGL. For example, IGF1 is involved in the proliferation of CGN progenitors (see above), but it also promotes the survival of CGNs, both in vitro and in vivo, by downregulating pro-apoptotic factors such as Bax, Bim, and Bad and upregulating Bcl-x(L) and Bcl-2 [20–22]. Also PACAP, which inhibits the proliferation of progenitors in the EGL [11], acts as a pro-survival factor for CGNs [23, 24]. Altogether, it seems that an orchestra of several extracellular factors plays an important, and complementary, role to glutamatergic inputs from mossy fibers in the control of CGN survival during their migration towards the IGL. Then, we examine by which molecular mechanisms NMDA and the pro-survival factors induce the survival of CGNs in the paradigm of KCl deprivation in cultured CGNs.

Molecular Pathways Related to the Neuroprotection of CGNs

It has been described that specific protein kinase cascades involving phosphoinosite 3-kinase (PI3K)/Akt, extracellular-signal regulated kinase (ERK), protein kinase A (PKA), or calcium/calmodulin kinase IV (CaMKIV) promote the neuroprotective activity of neurotransmitters and pro-survival factors by the increase of transcriptional activity. IGF1 exerts its neuroprotective effect through the activity of PI3K [25] and the phosphorylation of Akt [26]. The activation of PI3K/Akt decreases the activity of Forkhead transcription factors, favoring the survival role of IGF1 [22]. Other transcription factors, such as the family of myocite enhancer factor-2, also participate in IGF1-mediated survival effect [27] (Fig. 2).

Fig. 2
figure 2

Cellular mechanisms involved in the neuroprotection of CGNs. We introduced the signaling pathways involved in the survival of CGNs promoted by some neurotrophins, neuropeptides, and the NMDAR stimulation in the paradigm of potassium deprivation. Bold arrows indicate activation and dashed arrows denote inhibition

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The requirement of PI3K/Akt pathway was also observed in the neuroprotective effect of NMDA in immature [17] and mature [28, 29] CGNs. But this effect is not restricted to the activation of PI3K/Akt pathway as other protein kinases have also been involved. In mature CGNs, it has been showed that neuroprotection by NMDA is mediated by ERK activation [29]. However, other studies reported that inhibition of ERK pathway does not prevent the neuroprotective activity of NMDA [18, 28]. We recently reported that the survival role of PI3K/Akt and ERK pathways in CGNs depends on the small G proteins acting upstream [30]. We demonstrated that both pathways are involved in NMDA-mediated neuroprotection when they are activated by Ras. In contrast, the stimulation of the ERK pathway by other members of the small G proteins family, such as Rap1, is not sufficient to promote protection. Our results showed that the biological significance of NMDA-mediated activity of PI3K/Akt and ERK pathway depends on which monomeric-G protein is acting upstream. Furthermore, the requirement of Ras to activate these pathways also occurs for other protective factors [31]. Thus, these results support a central role for Ras in the survival of CGNs. NMDA-mediated activation of PI3K/Akt and ERK pathways stimulates the activation of CREB, an important transcription factor for the pro-survival effect of NMDA [32]. As indicated above, the neuroprotective effect of NMDA is mediated by the release of BDNF [33]. We recently described that the interaction of CREB with the Bdnf promoter is enhanced by potassium depolarization, whereas it is not improved by NMDA treatment [19]. We demonstrated that the stimulation of NMDAR triggers the CREB-dependent Nurr1 activation, which results in BDNF upregulation. Moreover, we have characterized Nurr1 as a key factor in NMDA-dependent survival of CGNs. Other factors mediating the survival of CGNs are related to the activation of ERK-CREB pathway. Interestingly, BMP6 has been described to promote CGN survival in culture through the stimulation of MEK-ERK-CREB pathway [34]. Thus, differential pathway activation by BMP6 could be related to its function promoting the formation of CGN progenitors (Smad-dependent) or the survival of CGNs (Smad-independent) during cerebellum development.

PACAP exerts a potent neuroprotective effect on cultured CGNs [35, 36]. More recently, it has shown in mice deficient for PAC1, the high-affinity PACAP receptor, that endogenous PACAP is crucial for the survival of CGNs [37]. The neuroprotective effect of PACAP is mediated through the activation of ERK [24] and the cAMP-dependent PKA [38, 39]. The increased activity of these pathways leads to the stimulation of pro-survival gene expression, such as c-fos or Bcl-2 [40, 41]. In addition, it has been described that PACAP mediates its neuroprotective effect, in part, by inhibition of delayed rectifier K(+) current (I(K)) via cAMP/PKA transduction pathway [42, 43]. Moreover, the survival effect of PACAP could be mediated by other pathways. It has been demonstrated that PACAP promotes the increase of intracellular calcium from intracellular stores and calcium influx through calcium channels [44, 45]. The mobilization of calcium allows the activation of CaMKIV and then the increase of CREB activity [46, 47]. More recently, it has been shown that PACAP-mediated protective effect is also through the release of tissue plasminogen activator [48].

The pivotal role of the Bcl-2 family proteins and the mitochondrial pathway in the apoptosis of CGNs has been extensively documented. Bax and other members of the Bcl-2 family are sufficient to promote apoptosis of CGNs [49, 50]. The reduction of these proteins is an important step in the neuroprotection of CGNs. It has been shown that neuroprotection mediated by trophic factors is associated with the reduction of Bax, Bad, and Bim levels [21, 22]. On the other hand, the increased expression of anti-apoptotic members of Bcl-2 family has been related to the neuroprotective effect of IGF1, PACAP, NMDA, and others [21, 41, 51]. The regulation of the Bcl-2 members allows a reduction of the apoptotic mitochondrial activity, reducing cytochrome c release [51, 52] and the activation of caspases [17, 21, 53, 54]. The inhibition of other apoptotic pathways is also an important step in the survival of CGNs. For instance, the inhibition of JNK prevents the apoptotic death of CGNs [17], and the long-lasting neuroprotective effect of NMDA was also related to the inhibition of JNK and the phosphorylation of c-jun [51]. Taken together, the survival of CGNs mediated by NMDAR and neurotrophin receptors results in the stimulation of common survival mechanisms and suppression of apoptotic pathways.

Conclusion

The data presented here support that CGNs are a key model to decipher which factors and cellular mechanisms underlie neuronal development and survival. Recent publications unraveled the action of numerous neurotrophic factors and stimulation of NMDAR in promoting the activity of CGNs at postnatal stages. Moreover, significant works revealed the similarity between these two, not only in the promotion of the survival, but also in terms of the signaling pathways that they activate in CGNs.