Elsevier

Neuroscience

Volume 273, 25 July 2014, Pages 79-91
Neuroscience

Distribution of fragile X mental retardation protein in the human auditory brainstem

https://doi.org/10.1016/j.neuroscience.2014.05.006Get rights and content

Highlights

  • FMRP is widely expressed in human brainstem neurons.

  • Nearly all bushy and octopus cells in the VCN are FMRP-immunoreactive.

  • FMRP is prominent in somata and dendritic arbors of the MSO.

  • FMRP is colocalized with the Kv3.1b potassium channel in MSO neurons and boutons.

  • Neurons in the human CN and SOC may be dysfunctional in FXS.

Abstract

Fragile X mental retardation protein (FMRP) binds select mRNAs, functions in intracellular transport of these mRNAs and represses their translation. FMRP is highly expressed in neurons and lack of FMRP has been shown to result in dendritic dysmorphology and altered synaptic function. FMRP is known to interact with mRNAs for the Kv3.1b potassium channel which is required for neurons to fire action potentials at high rates with remarkable temporal precision. Auditory brainstem neurons are known for remarkably high spike rates and expression of Kv3.1b potassium channels. Fragile X syndrome (FXS) is a genetic disorder caused by a mutation in the fragile X mental retardation 1 gene (Fmr1) resulting in decreased expression of FMRP and subsequent intellectual disability, seizures, attention deficit and hypersensitivity to auditory and other sensory stimuli. We therefore hypothesize that the auditory difficulties in FXS result, at least in part, from dysfunction of auditory brainstem neurons. To examine this hypothesis, we have studied normal human brainstem tissue with immunohistochemical techniques and confocal microscopy. Our results demonstrate that FMRP is widely expressed in cell bodies and dendritic arbors of neurons in the human cochlear nucleus and superior olivary complex and also that coincidence detector neurons of the medial superior olive colocalization of FMRP and Kv3.1b. We interpret these observations to suggest that the lower auditory brainstem is a potential site of dysfunction in FXS.

Introduction

Fragile X mental retardation protein (FMRP) is the product of the Fmr1 gene and is widely expressed in multiple tissues from the embryonic period into adulthood (Hinds et al., 1993). FMRP is an RNA-binding protein that functions mainly in activity-dependent translational regulation of a large number of mRNAs, including Kv3.1b and slack potassium channels, and is found both pre and post-synaptically (Akins et al., 2009, Akins et al., 2012, Strumbos et al., 2010, Zhang et al., 2012). Although, translation-independent actions of FMRP have recently been discovered (Brown et al., 2010, Zhang et al., 2012, Deng et al., 2013). In the adult, FMRP is widely expressed in epithelia [e.g. seminiferous tubules of the testis; esophagus] and nervous tissue (Hinds et al., 1993). In the brain, FMRP is expressed in neurons and glia throughout the brainstem, forebrain and cerebellum (Hinds et al., 1993, Feng et al., 1997, Wang et al., 2004, Jacobs et al., 2012), although not ubiquitously (Devys et al., 1993).

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability (Bassell and Warren, 2008) and results from a CGG triplet repeat expansion in the Fmr1 gene (Verkerk et al., 1991) and consequent repression of FMRP. FXS affects 1:3600 males and 1:8000 females (Cornish et al., 2008), is the most common genetic cause of autism (Bassell and Warren, 2008) and 15–30% of all FXS patients demonstrate autistic behaviors (Rogers et al., 2001, Hatton et al., 2006, Harris, 2011). Patients with FXS display cognitive disabilities, social deficits including language delays, seizures, autistic features, sensory hypersensitivity and hyperactivity (Eliez et al., 2001, Berry-Kravis, 2002, Hagerman et al., 2009). FXS is associated with a number of CNS dysmorphologies, including reduced volume of the cerebellar vermis, enlargement of the 4th ventricle (Mostofsky et al., 1998, Hoeft et al., 2010) and hypertrophy of the hippocampus (Kates et al., 1997) and caudate nucleus (Reiss et al., 1995, Eliez et al., 2001, Hoeft et al., 2010). Alterations in synaptic structure and function have been identified in FXS patients and animal models of FXS (e.g. Fmr1 knockouts [Fmr1 KO]; Pfeiffer and Huber, 2009) and a number of presynaptic and postsynaptic proteins have abnormal levels in Fmr1 KO (Li et al., 2002, Klemmer et al., 2011). Furthermore, in FXS and Fmr1 KO there is a high density of immature dendritic spines (human – Rudelli et al., 1985, Hinton et al., 1991, Comery et al., 1997, Irwin et al., 2000; mouse – Nimchinsky et al., 2001, Galvez et al., 2003). Cultured hippocampal neurons from Fmr1 KO mice give rise to shorter dendrites with fewer dendritic spines compared to controls (Braun and Segal, 2000, Castren et al., 2005) and FMRP-deficient mice have abnormally arranged dendritic fields in the somatosensory cortex (Galvez et al., 2003), more primary dendrites in the olfactory bulbs (Galvez et al., 2005) and spinal motor neurons with immature dendritic arbors (Thomas et al., 2008). Fmr1 KO flies also demonstrate significant overgrowth of dendrites and axons (Zarnescu et al., 2005). These results, taken together, suggest that FMRP plays a role essential to normal maturation and function of the central nervous system.

In our previous post-mortem studies of the autistic brain, we examined the brainstem of a 32-year-old male diagnosed with autism and FXS (Kulesza and Mangunay, 2008). In this case, we observed neurons in the medial superior olive (MSO), a prominent brainstem nucleus which contains coincidence detector neurons that function in sound source localization and encoding temporal features of sound, to be significantly smaller (a nearly 50% reduction in cell body area) and significantly more round (i.e. immature) compared to an age-matched control (Kulesza and Mangunay, 2008). Moreover, there was significantly more variability in the orientation of these MSO neurons in the FXS/autism brain compared to an age-matched control. In addition, we have demonstrated that FMRP is highly expressed in brainstem coincidence detector neurons across species including the nucleus laminaris of alligator and chicken and the MSO in gerbils and human (Wang et al., 2013). Together, we interpret the dysmorphology of MSO neurons in FXS and the abundance of FMRP in the MSO to suggest that auditory-processing deficits in FXS result, at least in part, from dysfunction of brainstem centers. Further, it is believed that the cell types which express FMRP are the most severely impacted in FXS (Hinds et al., 1993) and we hypothesize that FMRP is widely expressed in neuronal cell bodies and dendritic arbors in the cochlear nuclei and superior olivary complex (SOC). Additionally, FMRP is known to play an important role in activity-dependent regulation and tonotopic expression of the Kv3.1b potassium channel in the auditory brainstem and the tonotopic gradient of Kv3.1b expression is required for accurate coding of complex sounds (Strumbos et al., 2010). To examine the distribution of FMRP in the human auditory brainstem and explore possible functional deficits in FXS, we have used immunohistochemistry and confocal microscopy to map the distribution of FMRP in control human dorsal and ventral cochlear nuclei (DCN and VCN) and SOC. Furthermore, we have examined the colocalization of FMRP and Kv3.1b in the MSO.

Section snippets

Tissue sectioning

This report is based on the examination of brainstems from seven individuals ranging in age from 57 to 96 years of age (average 78.6 ± 5.9 years; five female/two male). Table 1 shows the age, cause of death and post-mortem interval for specimens used in this study. All specimens were obtained with permission from the PA Humanities Gifts Registry. Brainstems were only included in this study if they met the following criteria: (1) the cause of death was not neurological, (2) there were no signs of

General brainstem distributions

In order to obtain a more global understanding of the neuronal distribution of FMRP in the human brainstem, we estimated the number of FMRP+ neurons in nuclei along the rostro-caudal length of the brainstem. Specifically, we examined FMRP expression in the principal nucleus of the inferior olive (IO), facial nucleus (FN), MSO, pontine nuclei (PN) and the abducens nucleus (AbN). We found that the vast majority of neurons in the FN and MSO were FMRP+ (Fig. 1; 85 ± 9% and 82 ± 9%, respectively; mean ± 

Discussion

To the best of the authors’ knowledge, this is the first quantitative investigation of FMRP-immunolabeling in the human brainstem and the first characterization of FMRP+ neurons in the human cochlear nucleus and SOC. We have identified FMRP+ neurons in each of the brainstem nuclei we investigated, but the proportion varied considerably by nucleus. Specifically, we found that nearly 85% of FN neurons but only about 13% of neurons in the principal nucleus of the IO were FMRP+. We interpret these

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported in part by a grant from the Lake Erie Consortium for Osteopathic Medical Training. The authors would like to thank Jerome McGraw, Kristen Ruby and George Grignol for technical assistance.

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