BMP Signaling in Axon Regeneration

Abstract

Neuronal competence to re-extend axons and a permissive environment that allows growth cone navigation are two major determinants for successful axon regeneration. Here, we review the roles of bone morphogenetic protein (BMP) signaling in mediating both neuronal and glial injury responses after CNS injury. BMPs can activate a proregenerative transcription program in neurons through Smad-mediated canonical pathway, or act locally on cytoskeleton assembly at distal axons via non-canonical pathways. Emerging evidence implicates retrograde BMP signalosomes in connecting the cytoskeletal and nuclear responses. In addition, BMP/Smad signaling modulates neurotrophin-mediated axonal outgrowth, and interacts with the epigenetic machinery to initiate epigenetic reprogramming for axon regeneration. Besides their influences on neurons, BMPs also regulate astrogliosis, inflammatory processes, and neural progenitor cell differentiation at the injury site, all of which can either positively or negatively modify the injury microenvironment. Lastly, an increasing number of BMP signaling partners, sensitizers, and downstream effectors collectively fine-tune the signaling intensity and spatiotemporal dynamics of BMP activity in an integrated signaling network during axon regeneration.

Introduction

Mature neurons in the CNS regenerate their injured axons minimally, owing to an age-dependent decline of axon growth potential [1] and an inhibitory environment [2]. Inactivation of the external inhibitory molecules is insufficient for long-distance axon regeneration, as demonstrated in studies on functional interference with myelin-based inhibitors [3] or chondroitin sulfate proteoglycans (CSPGs) [4]. On the other hand, enhancing the intrinsic axon growth potential of adult neurons by itself also results in modest regeneration [1]. Hence, successful regenerative strategies need to address both intrinsic and extrinsic hurdles. Bone morphogenetic proteins (BMPs) have emerged as an important class of signaling molecules that mediate both neuronal axon growth potential and glial injury responses.

BMPs are members of the TGFβ superfamily that signal through serine/threonine kinase receptors to activate Smad family transcription factors through C-terminal phosphorylation (pSmad^C) [5]. Smad1, -5, and -8 isoforms mediate canonical signaling of the BMP family, while Smad2 and -3 mediate signaling of the TGFβ family. In addition to the pSmad^C-mediated transcriptional program, BMPs can also activate non-canonical, Smad-independent pathways that include LIM kinase (LIMK), p38/MAPK, phosphatidylinositol 3-kinase (PI3K), and Rho-like small GTPases. Some of the known functions of these pathways involve local cytoskeletal dynamics by way of modulating the actin depolymerization factor cofilin. Additionally, BMP co-receptors and signaling sensitizers further increase the functional complexity of BMPs in the CNS (for an in-depth review on BMP pathways in the nervous system, please refer to [6]). Here, we discuss the involvement of both the canonical and non-canonical BMP signaling in coordinating neuronal and glial injury responses, focusing on those that influence the outcome of axon regeneration after CNS injury.

BMP signaling mediates axon growth potential

To date, BMPs have not been directly linked to developmental axon elongation in vivo. This could be due to their functional redundancy (the BMP family consists of 20 members) [7], or to the early embryonic lethality of currently available mouse knockout lines. The development of more sophisticated conditional mouse models may yield conclusive evidence regarding the role of BMP signaling in axon development. In primary neuron cultures, BMPs function as growth factors to stimulate axon growth in developing striatal GABAergic neurons [8], raphe serotonergic neurons [9], cerebellar granule neurons [10], retinal ganglion cells (RGCs) [11], and spinal motor neurons (SMNs) [12]. BMP also interacts with neurotrophin signaling as part of a tightly regulated signaling network to ensure proper axon development. For instance, in developing DRG sensory neurons, neurotrophins robustly promote axonal outgrowth through the RAF-MEK kinase cascade [13]. Conditional activation of B-RAF kinase promotes both developmental and regenerative axon growth [14]. BMP signaling is required for neurotrophin-mediated axonal outgrowth in DRG neurons, by way of transcriptional regulation of two effectors of RAF-MEK kinase signaling, Erk1 and Erk2, thereby sensitizing neurotrophin responsiveness [15]. In vivo, abrogation of NGF or RAF-MAP kinase signaling results in axonal terminal branching defect [13], which is phenocopied by conditional knockout of Smad1 in neural-crest derived cells, including DRG neurons [15]. In addition, BMP signaling potentiates NGF induction of the transcription factor Egr [16]. BMPs also potentiate neurotrophin 3-induced neurite outgrowth of peripheral neurons [17]. On the other hand, BMP4 has been shown to function as a target-derived cue that limits the number of sensory neurons and the extent of terminal peripheral nerve innervation [18]. In developing trigeminal ganglia (TG) sensory neurons, a forward genetic screen has identified Megf8, a large putative transmembrane protein, as a modifier of BMP4 signaling that inhibits axon growth [19]. Interestingly, in TG sensory neurons, retrograde BMP signaling regulates gene expression and patterning along the dorsoventral axis of the TG [20]. Furthermore, BMP7 in the roof plate of spinal cord functions to repel axons of developing commissural neurons [21]. Taken together, BMPs mediate axon development in a cell-type specific and context dependent manner. For a full understanding, one must consider which signaling cascade is activated by BMPs, in what neuronal subtypes, in which subcellular compartment, and at what developmental stages.

Initial evidence for a pro-regenerative role of BMP-Smad signaling came from studies of the conditioning lesion paradigm, where a prior peripheral axotomy of DRG neurons drastically increases axon growth potential of the centrally projecting axons that are usually refractory to regeneration [22]. Smad1 is induced after a conditioning lesion, and its activation is required for enhanced axon growth potential. In contrast, a central axotomy fails to induce or activate Smad1 [23]. In a followup study, we designed an intrathecal injection of an adeno-associated virus that encodes BMP4 (AAV-BMP4) to specifically target DRG neurons. This led to nuclear accumulation of activated Smad1, promotion of axon growth potential and sensory axon regeneration in a mouse model of SCI [24]. Notably, Smad1 is developmentally regulated, with robust expression in developing DRG neurons. Aside from regulating Erk1 and Erk2 transcription by pSmad1^C, Smad1 can also respond to neurotrophin stimulation via linker phosphorylation (pSmad1^L). pSmad1^L takes part in a negative feedback mechanism to prevent axonal overgrowth in the target field by upregulating DUSP6, an Erk-specific dual-specificity phosphatase, leading to reduced pErk1/2. Hence, through differential phosphorylation, Smad1 helps to shape sensory axon development by converging neurotrophin and BMP signaling to maintain a balanced ratio of Erk1/2 and pErk1/2 [15]. In further support for a positive role of BMP-Smad signaling in axon regeneration, acute knockdown of Smad1 hinders axonal regeneration in a sciatic crush model in vivo [25]. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibits neurite outgrowth in DRG explants and retards early axonal regeneration in injured sciatic nerve. It is worth mentioning that Noggin is downregulated in DRGs after a conditioning lesion [26].

Expression profiling of regeneration-associated genes (RAGs) showed that Sprr1a, Npy, Galanin and to a lesser extent, Vip, are downregulated in DRGs with Smad1 ablation. Chromatin immunoprecipation (ChIP) studies confirmed specific binding of Smad1 at the promoters of target genes. Our recent studies also demonstrate that Smad1 helps to recruit histone-modifying enzymes such as histone acetylases (HATs) and dissipate histone deacetylases (HDACs), leading to increased histone H4 acetylation (AcH4); AcH4 enrichment in turn facilitates Smad1 promoter occupancy. It turns out that diminished axon growth potential is associated with histone hypoacetylation, whereas a conditioning lesion can restore histone acetylation levels [27, 28]. Importantly, pharmacological inhibition of HDACs is capable of initiating epigenetic reprogramming, leading to induction of multiple RAGs and promotion of sensory axon regeneration after SCI. Together, these studies identify a pro-regenerative transcriptional module, consisting of Smad1 and histone enzymes for RAG regulation [28]. Consistently, Smad1 is among the differentially upregulated genes in axon-sprouting vs. non-sprouting neurons after cerebral ischemia [29]. Moreover, in injured hypoglossal motor neurons, Smad1, 2, and 4 are upregulated [30], and in RGC, BMP-Smad signaling promotes survival after NMDA induced retina damage [31]. However, BMPs' role in axon regeneration in other classes of neurons awaits future studies.

BMP signaling regulates cytoskeletal dynamics during axon development

BMP signaling can act locally to mediate cytosketetal assembly in distal axonal or dendritic compartments, through Smad-independent, non-canonical pathways. For instance, LIMK can be activated by binding to type II BMPR and regulates BMP-dependent dendritogenesis [32]. On the other hand, in cerebellar granule neurons, BMP2 inhibits neurite outgrowth by a LIMK-dependent mechanism [33]. BMP7 has been shown to induce growth cone turning via local actin dynamics through ADF/cofilin activity controlled by LIMK and slingshot phosphatase [34]. In Drosophila motor neurons, both local LIMK-mediated and retrograde BMP signaling are involved in synaptic formation and stability at the neuromuscular junction (NMJ) [35, 36]. Whether modulation of the BMP-LIMK-ADF/Cofilin pathway could enhance axon regeneration needs to be tested.

Aside from the LIMK/cofilin pathway, the glycosylphosphatidylinositol (GPI)-anchored repulsive guidance molecules (RGMs) have been identified as BMP co-receptors as they can form a complex with BMP receptors [37, 38]. RGMb (Dragon) is highly expressed in adult DRG sensory neurons and promotes neurite outgrowth by sensitizing BMP signaling [26]. RGMb deletion decreases BMP signaling and inhibits early axonal regeneration in the sciatic nerve crush model [26]. In contrast, RGMa causes, by a BMPR-independent mechanism, growth cone collapse and neurite outgrowth inhibition by engaging the transmembrane receptor neogenin to activate the small GTPase RhoA and its downstream effectors Rho kinase and PKC [39]. Intrathecal administration of a blocking antibody against RGMa enhances CST regeneration and improves functional recovery [40]. It is also noteworthy that BMP can directly bind to neogenin, leading to RhoA activation and a dampening effect on Smad in non-neuronal cell culture studies [41]. The relevance of the BMP-RGM or BMP-neogenin signaling in axon regeneration in vivo has not been determined.

Intra-axonal BMP signalosome trafficking

Long-range, retrograde signaling of target-derived BMPs helps to shape neural circuit assembly [42]. The effects of target-derived BMP4 and BDNF are interconnected: BDNF induces translation of axonally localized Smad1/5/8 transcripts, and the axon-derived Smad1/5/8 is then translocated to the soma, where it is activated by BMP-induced signaling endosomes [43].

BMP signaling is also involved in axonal transport and organization of the microtubule network in developing motor neurons in Drosophila [44]. Endocytosis of BMP receptors and dynein-dependent retrograde transport of BMP signalosome along the axon have been demonstrated [45]. In zebrafish SMN axons, type I BMPR is observed in punctate vesicular structures distributed along the neurite, and BMP signalosome trafficking, mediated by atlastin, a membrane-bound GTPase linked to cases of hereditary spastic paraglegia (HSP), has been implicated in microtubule dynamics [46]. Knockdown of atlastin leads to enhanced BMP signaling and aberrant axonal branching due to lack of stable microtubules. It is conceivable that intra-axonal BMP receptor trafficking may affect axon regeneration by regulating microtubule stability and by coupling the cytoskeletal dynamics at the distal axon with the nuclear transcriptional response.

Studies using microfluidic chambers to separately manipulate BMP signaling in the soma vs. axon terminal may shed light on this hypothesis.

BMP signaling regulates glial scar formation

One of the major differences between axon regeneration after injury and developmental axon growth is the interference from glial scarring after CNS injury. While the dense glial scar that builds up over time is detrimental to axon regeneration due to increased deposition of CSPGs [47], the early hypertrophic astroglial response is thought to be beneficial for neural repair by restricting scar size [48]. Engraftment of astrocytes derived from glial-restricted embryonic precursors treated with BMP4 after SCI promotes axon regeneration and functional recovery [49].

The local injury microenvironment favors astrocyte differentiation as illustrated by the preferential differentiation of engrafted pluripotent embryonic neural precursor cells (NPCs) into astrocytes in the injured spinal cord [50]. After SCI, a number of BMPs are upregulated in local neurons, astrocytes, oligodendrocytes, microglia/macrophages, and endogenous NPCs in the spinal cord, with different temporal patterns [51, 52]. BMPs are thought to restrict NPCs to the astroglial lineage while suppressing the neuronal and oligodendroglial differentiation, similar to their effect in neurosphere cultures derived from adult mouse spinal cord [53]. In cultured astrocytes, BMPs increase CSPG expression, in line with the notion that BMPs drives gliogenesis and glial scar formation, which has also been demonstrated in a demyelinating spinal cord lesion model [54]. However, global inhibition of BMP signaling in vivo at the SCI site yields conflicting results. In one study, transplantation of fetal NPCs engineered to express Noggin promoted neuronal and oligodendrocyte differentiation while suppressing generation of astrocytes, along with functional recovery after SCI [55]; but in another study, astroglial differentiation was unaffected, but instead, it led to an increase in both lesion volume and the number of infiltrating macrophages [56]. Intrathecal delivery of Noggin also gave rise to conflicting results: it enhanced locomotor activity and CST axons regeneration in one study [57], but failed to attenuate GFAP expression in another [53]. These discrepancies are attributed to different grafted cells, various injury models (compression vs. contusion and ischemia), and use of cyclosporine A as immunosuppressor in some studies, which may mask the effect of Noggin. Another contributing factor may be the varying ratio of BMPR isoform expression in different glial cells or NPCs. BMPR1a and -1b exert opposing effects, with BMPR1a positively, and BMPR1b negatively regulating astrogliosis and wound closure after SCI [58]. Conditional knockout of BMPR1a in GFAP-expressing cells results in defective astrocytic hypertrophy, increased inflammatory cell infiltration, in conjunction with reduced axonal density and deficits in locomotor recovery after severe SCI. Notably, these effects of BMPR1a are independent of nuclear Smad signaling. In contrast, BMPR1b deletion leads to hyperactive reactive astrocytes and reduced lesion volume. Loss of both BMPR isotypes resulted in normal-appearing reactive astrocytes [58].

BMP also promotes astrocyte differentiation from oligodendrocyte precursor cells (OPCs) at the expense of oligodendrocytes [59]. This occurs through upregulation of Id2 and Id4, which in turn interact with Olig1 and Olig2 [60]. Furthermore, reactive astrocytes in the contused spinal cord themselves secrete BMP, which further limits oligodendrocyte differentiation from OPCs [61]. BMPs also have immunomodulatory roles. In an in vitro co-culture of cortical explants with primary microglia, p38 MAPK is activated in primary microglia after axonal transection and is required for phagocytic activity of microglia to engulf axon debris [62].

TGFβ is another important activator of astrogliosis through the Smad pathway [63]. Upon CNS injury, fibrinogen leaks out of injured vasculature and functions as a carrier of a latent form of TGFβ, which is then activated by astrocytes, leading to Smad2 phosphorylation and glial scarring [64]. On the other hand, microtubule stabilization by Taxol treatment reduces scar formation and promotes axonal regeneration after SCI [65]. The decreased CSPG in Taxol-treated animals is ascribed to decreased nuclear translocation of Smads, leading to dampening of the TGFβ signaling. These results raise the possibility of an involvement of fibrinogen or microtubule-Smad interaction in BMP-mediated astrogliosis.

Conclusion

As in developmental processes, BMPs exert a wide range of influences over both neurons and glia after CNS injury. BMPs can activate a pro-regenerative transcription program in neurons through Smad-mediated canonical pathway, or act locally on cytoskeletal dynamics at the growth cone via non-canonical pathways. The responses of the two subcellular compartments to BMPs may be connected via intra-axonal BMP signalosome trafficking. BMPs also function as an injury signal to coordinate glial responses and neural progenitor cell differentiation at the injury site. Establishing a comprehensive view of the multifaceted roles of BMP signaling after CNS injury is a prerequisite to design BMP-based therapeutic strategies to promote axonal repair and functional recovery.

Figure 1.

Crosstalk of BMP and neurotrophin signaling in neurons. Through differential phosphorylation, Smad1 serves as a converging node to maintain a balanced ratio of ERK/pERK. BMPs signal through C-terminal phosphorylation of Smad1, which then makes nuclear entry and regulates transcription of downstream targets, such as Erk1 and Erk2. Neurotrophin stimulation leads to ERK/MAPK-mediated linker phosphorylation of Smad1, which in turn upregulates DUSP6, an ERK-specific phosphatase, thereby reducing pErk levels and constituting a negative feedback loop. PI3K signaling can induce Smad1, while GSK-mediated linker phosphorylation of Smad1 marks it for proteasome degradation via Smurf1-dependent polyubiquitination pathway. BDNF can also stimulate intra-axonal translation of Smads, which are then translocated to soma to be activated by BMP signalosomes, thereby connecting retrograde signaling of BDNF and BMP.

Figure 2.

Schematic of BMP signaling in promoting axon regeneration. BMP can activate a pro-regenerative transcriptional program through the Smad-mediated canonical pathway. Smad1 works together with histone-modifying enzymes to increase histone acetylation levels, which in turn facilitates Smad1 promoter binding and transcription of regeneration-associated genes (RAGs) by way of local chromatin relaxation. BMP also regulates local cytoskeletal assembly through non-canonical pathways, such as LIMK-cofilin signaling. Retrogradely transported signaling endosomes may control microtubule stability and link cytoskeletal and nuclear responses to BMPs.

Figure 3.

Schematic of BMP signaling in neural progenitor cell differentiation, gliogenesis and scar formation after CNS injury. Astroglial activation plays a dual role: restricting scar size, but also hindering axon regeneration via CSPG deposition. BMPR1a positively and BMPR1b negatively regulates astrocytosis. BMP also drives astroglial differentiation of OPCs, and BMP secreted by reactive astrocytes further limits oligodendrocyte differentiation.

Highlights.

1. BMP regulates axon growth potential via Smad1-mediated canonical signaling.

2. BMP mediates cytoskeletal assembly in distal axon via non-canonical pathways.

3. Retrograde signalosomes couple cytoskeletal and nuclear effects of BMP.

4. BMP coordinates neuronal and glial injury responses after CNS injury.