Posttranscriptional regulation of gene expression is increasingly recognized as an important general mechanism by which the function and morphological plasticity of dendrites, axons, and synapses are controlled locally in response to various environmental signals (44
). Our findings reveal pivotal roles for miR-375 and HuD, two different molecules that bind mRNAs encoding neuron-specific proteins, in the regulation of neurite outgrowth and differentiation in cultured neuronal cells and hippocampal granule neurons in vivo
. There is an inverse relationship between the levels of expression of miR-375 and HuD during mouse brain development: the levels of miR-375 are highest in early embryos and decrease by E16, whereas HuD levels are low in early embryos and increase markedly by E16, when neurogenesis peaks (Fig. ). We showed that by binding to the HuD 3′ UTR, miR-375 impedes the translation of HuD, thereby suppressing the expression of multiple HuD target mRNAs that encode neuron-specific proteins. By acting in an antagonistic manner to regulate the stability and translation of proteins involved in neurite growth and in the stability of dendrites and synapses, miR-375 and HuD may control the formation, maintenance, and plasticity of neuronal circuits in the mammalian brain. Consistent with such roles for miR-375 and HuD, we found that overexpression of miR-375 or silencing of HuD abolished the ability of BDNF to stimulate neurite growth. In mature neurons, BDNF is critical for maintaining neuronal plasticity and neuronal outgrowth, but BDNF also has many developmental roles, including neuronal differentiation (17
). We found that miR-375 disrupts BDNF-mediated neurite outgrowth in neuroblastoma cells (Fig. ), indicating that miR-375 and HuD can interact at the posttranscriptional level with BDNF signaling pathways that control neuronal morphology and function. Therefore, we propose that miR-375 inhibits premature neuronal differentiation and might help to maintain a proliferative phenotype of embryonic neural stem cells and progenitors. Ongoing efforts are aimed at examining the role of miR-375 in the developmental regulation of neuronal differentiation.
Our findings suggest that in differentiated neurons, low miR-375 abundance is critical for maintaining elevated HuD levels. In turn, HuD helps to keep a constitutive density of neurites in mouse hippocampus and to elicit de novo neurite outgrowth after BDNF treatment of cultured cells. miR-375 interacts with a specific HuD 3′ UTR site that was strikingly well conserved among rodent and human genes, despite the generally poor conservation of the 3′ UTRs. miR-375 lowered both HuD mRNA stability and translation; the resulting diminution of HuD levels reduced the expression of critical proteins implicated in neuronal signaling, survival, and cytoskeletal organization. The inhibition of neurite outgrowth by miR-375 was rescued by HuD overexpression from an HuD mRNA that lacks the miR-375 site.
Recent en masse analysis of HuD-associated mRNAs in mouse brain revealed that many HuD targets encoded proteins with vital roles in neuronal differentiation, cytoskeletal transport, and RNA metabolism (16
), including many of the targets we identified here using human BE(2)-M17 cells. These findings fully support HuD's ability to promote neuronal development, synaptic plasticity, and nerve regeneration (reviewed in references 37
). Accordingly, ectopic interventions to overexpress or downregulate HuD in cultured neuronal models revealed a role for HuD in the expression of target mRNAs and in controlling neuronal morphology (19
). HuD-null mice show deficient neurogenesis, nerve development, and motor and sensory functions (4
), while HuD-overexpressing mice displayed alteration in hippocampus physiology associated with impaired learning and memory (14
With neuronal abnormalities arising from both elevations and reductions in HuD abundance, it is surprising that the mechanisms responsible for regulating HuD expression and function were largely unknown until now. In this regard, the discovery that miR-375 represses HuD expression provides a means to express high HuD levels in tissues with low miR-375 (e.g., brain) and low HuD levels in tissues that express miR-375 in abundance (e.g., heart and muscle). It should be noted that while HuD was previously believed to be strictly neuronal, it was detectable in other tissues, like testis, pituitary gland, and lung (Fig. ), suggesting that HuD could have functions in cells other than neurons. As individual- and species-specific variations appear to exist, a deeper analysis of HuD tissue distribution is warranted. Regarding expression and function, HuD was shown to be methylated by the coactivator-associated arginine methyltransferase 1 (CARM1) on Arg 236, a modification that reduces its ability to interact with a target transcript, the p21Waf1
). HuD was also found to bind the HuD mRNA (16
), suggesting a possible self-regulatory loop akin to those shown for other RBPs, like TTP, AUF1, and HuR (11
). In models of nerve regeneration and contextual learning, HuD abundance was found to increase, but the mechanisms responsible were not elucidated (6
). In the rat, HuD protein levels were highest at ~E16, declined perinatally, and remained low in the adult rat brain (25
), precisely the opposite of the expression pattern that was observed for miR-375 in the developing mouse telencephalon (Fig. ). Thus, the finding reported here that miR-375 regulates HuD production provides a mechanism to explain the magnitude and tissue-specific abundance of HuD.
Analyzed as a group, neuronal elav/Hu proteins were shown to be phosphorylated by protein kinase C (PKC); this modification changes their distribution and interaction with GAP-43 mRNA, but the specific changes in HuD were not studied separately from the other neuronal elav/Hu proteins (38
). Interestingly, exposure of cultured SH-SY5Y to Aβ(1-42), the product of APP (amyloid precursor protein) cleavage, strongly lowered the interaction of neuronal elav/Hu proteins with ADAM10 (a d
etalloproteinase 10), an integral membrane protein that acts as an α-secretase and thereby promotes the nonamyloidogenic processing of APP (5
). Whether HuD specifically mediated this process and which precise mechanisms (transcriptional and/or posttranscriptional) control ADAM10 expression also remain to be investigated.
In mammalian cells, posttranscriptional gene regulation by mRNA-interacting factors is recognized as a major mechanism for determining the amount of protein expressed. Besides governing the amount and timing of the proteins expressed, mRNA-binding factors can effectively govern the location where the protein is synthesized. This feature is particularly relevant to the neuronal system, given the special morphology of neurons, where mRNAs are synthesized in the nucleus but the gene products are often required at the distal ends of dendrites and axons, far from the nucleus. The elucidation of the mechanisms that govern the transport and local translation of mRNAs in neuronal cells has become an area of intense research. Several RBPs have been implicated in neuronal mRNA transport, stability, and localized translation, including elav/Hu, AUF1, cytoplasmic polyadenylation element binding (CPEB), Musashi, zipcode-binding protein (ZBP1 and -2), K homology-type splicing regulatory protein (KSRP), NOVA-1 and -2, fragile X mental retardation protein (FMRP), and Staufen (Stau1 and -2) (reviewed by Bolognani and Perrone-Bizzozero [15
]). A growing number of microRNAs have also been implicated in neuronal differentiation, integrity, and plasticity (miR-124a and miR-133b); in the formation of synapses in postmitotic neurons (miR-134 and miR-9a); and in circadian rhythms (miR-132 and miR-219) (reviewed by Gao [22
]). In this context, maintenance of low miR-375 levels allows the expression of high levels of HuD, which can then coordinate the production of proteins that establish, restore, and maintain neuronal function. Since restoring HuD expression did not totally rescue the impairment in neuritogenesis (Fig. ), miR-375 likely controls the expression of important neuronal proteins in addition to HuD. Further study of miR-375-regulated targets will be important in order to fully understand how miR-375 coordinates dendrite formation and maintenance.
In conclusion, the finding that a single microRNA regulates expression of an RBP with a pivotal function in neurons represents an effective mechanism of “amplification” of a gene expression program and highlights the richness and complexity of posttranscriptional gene regulation in the nervous system. RBPs and microRNAs are increasingly recognized as vital mediators of gene expression in neuronal cells, as they tightly regulate the timing and specific compartment in which particular proteins are expressed. Additional examples of interplay between microRNAs and RBPs regulating neuronal gene expression are bound to emerge as we gain deeper understanding of the magnitude, timing, and spatial regulation of neuronal proteins expressed in response to endogenous and environmental signals.