Delphine Bouhy

The hnRNP family: insights into their role in health and disease

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs) that contribute to multiple aspects of nucleic acid metabolism including alternative splicing, mRNA stabilization, and transcriptional and translational regulation. Many hnRNPs share general features, but differ in domain composition and functional properties. This review will discuss the current knowledge about the different hnRNP family members, focusing on their structural and functional divergence. Additionally, we will highlight their involvement in neurodegenerative diseases and cancer, and the potential to develop RNA-based therapies.

Neurofilament phosphorylation and their proline-directed kinases in health and disease

The intermediate filaments called neurofilaments (NFs) are the main cytoskeleton elements in neurons. They are mainly present in the adult axonal cytoskeleton, where they are extensively phosphorylated and their phosphorylation status is pivotal for their properties and functions. Specific phosphorylation of the C‐terminal domains of the large NF subunits has been implicated in radial axonal growth, NF bundling, and NF axonal transport. Many kinases and phosphatases are involved in regulating the NF phosphorylation status, and this complex interplay is of growing interest as hyperphosphorylation of NFs is a hallmark of several neurodegenerative diseases. In this review, we focus on the in vivo relevance of C‐tail phosphorylation of the large NF subunits and give an overview of the kinases and phosphatases involved in regulating the phosphorylation status of the NFs.

Charcot–Marie–Tooth causing HSPB1 mutations increase Cdk5-mediated phosphorylation of neurofilaments

Mutations in the small heat shock protein HSPB1 (HSP27) are a cause of axonal Charcot–Marie–Tooth neuropathy (CMT2F) and distal hereditary motor neuropathy. To better understand the effect of mutations in HSPB1 on the neuronal cytoskeleton, we stably transduced neuronal cells with wild-type and mutant HSPB1 and investigated axonal transport of neurofilaments (NFs). We observed that mutant HSPB1 affected the binding of NFs to the anterograde motor protein kinesin, reducing anterograde transport of NFs. These deficits were associated with an increased phosphorylation of NFs and cyclin-dependent kinase Cdk5. As Cdk5 mediates NF phosphorylation, inhibition of Cdk5/p35 restored NF phosphorylation level, as well as NF binding to kinesin in mutant HSPB1 neuronal cells. Altogether, we demonstrate that HSPB1 mutations induce hyperphosphorylation of NFs through Cdk5 and reduce anterograde transport of NFs.

Animal models and therapeutic prospects for Charcot–Marie–Tooth disease

Charcot–Marie–Tooth (CMT) neuropathies are inherited neuromuscular disorders caused by a length‐dependent neurodegeneration of peripheral nerves. More than 900 mutations in 60 different genes are causative of the neuropathy. Despite significant progress in therapeutic strategies, the disease remains incurable. The increasing number of genes linked to the disease, and their considerable clinical and genetic heterogeneity render the development of these strategies particularly challenging. In this context, cellular and animals models provide powerful tools. Efficient motor and sensory tests have been developed to assess the behavioral phenotype in transgenic animal models (rodent and fly). When these models reproduce a phenotype comparable to CMT, they allow therapeutic approaches and the discovery of modifiers and biomarkers. In this review, we describe the most convincing transgenic rodent and fly models of CMT and how they can lead to clinical trial. We also discuss the challenges that the research, the clinic, and the pharmaceutical industry will face in developing efficient and accessible treatment for CMT patients. Ann Neurol 2013;74:391–396

A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot–Marie–Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.

Mutant HSPB1 causes loss of translational repression by binding to PCBP1, an RNA binding protein with a possible role in neurodegenerative disease

The small heat shock protein HSPB1 (Hsp27) is an ubiquitously expressed molecular chaperone able to regulate various cellular functions like actin dynamics, oxidative stress regulation and anti-apoptosis. So far disease causing mutations in HSPB1 have been associated with neurodegenerative diseases such as distal hereditary motor neuropathy, Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis. Most mutations in HSPB1 target its highly conserved α-crystallin domain, while other mutations affect the C- or N-terminal regions or its promotor. Mutations inside the α-crystallin domain have been shown to enhance the chaperone activity of HSPB1 and increase the binding to client proteins. However, the HSPB1-P182L mutation, located outside and downstream of the α-crystallin domain, behaves differently. This specific HSPB1 mutation results in a severe neuropathy phenotype affecting exclusively the motor neurons of the peripheral nervous system. We identified that the HSPB1-P182L mutant protein has a specifically increased interaction with the RNA binding protein poly(C)binding protein 1 (PCBP1) and results in a reduction of its translational repressive activity. RNA immunoprecipitation followed by RNA sequencing on mouse brain lead to the identification of PCBP1 mRNA targets. These targets contain larger 3′- and 5′-UTRs than average and are enriched in an RNA motif consisting of the CTCCTCCTCCTCC consensus sequence. Interestingly, next to the clear presence of neuronal transcripts among the identified PCBP1 targets we identified known genes associated with hereditary peripheral neuropathies and hereditary spastic paraplegias. We therefore conclude that HSPB1 can mediate translational repression through interaction with an RNA binding protein further supporting its role in neurodegenerative disease.