New pathologic mechanisms in nucleotide repeat expansion disorders.

Tandem microsatellite repeats are common throughout the human genome and intrinsically unstable, exhibiting expansions and contractions both somatically and across generations. Instability in a small subset of these repeats are currently linked to human disease, although recent findings suggest more disease-causing repeats await discovery. These nucleotide repeat expansion disorders (NREDs) primarily affect the nervous system and commonly lead to neurodegeneration through toxic protein gain-of-function, protein loss-of-function, and toxic RNA gain-of-function mechanisms. However, the lines between these categories have blurred with recent findings of unconventional Repeat Associated Non-AUG (RAN) translation from putatively non-coding regions of the genome. Here we review two emerging topics in NREDs: 1) The mechanisms by which RAN translation occurs and its role in disease pathogenesis and 2) How nucleotide repeats as RNA and translated proteins influence liquid-liquid phase separation, membraneless organelle dynamics, and nucleocytoplasmic transport. We examine these topics with a particular eye on two repeats: the CGG repeat expansion responsible for Fragile X syndrome and Fragile X-associated Tremor Ataxia Syndrome (FXTAS) and the intronic GGGGCC repeat expansion in C9orf72, the most common inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Our thesis is that these emerging disease mechanisms can inform a broader understanding of the native roles of microsatellites in cellular function and that aberrations in these native processes provide clues to novel therapeutic strategies for these currently untreatable disorders.

Impaired sensorimotor gating in Fmr1 knock out and Fragile X premutation model mice.

Fragile X syndrome (FXS) is a common inherited cause of intellectual disability that results from a CGG repeat expansion in the FMR1 gene. Large repeat expansions trigger both transcriptional and translational suppression of Fragile X protein (FMRP) production. Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is an allelic neurodegenerative disease caused by smaller "pre-mutation" CGG repeat expansions that enhance FMR1 transcription but lead to translational inefficiency and reduced FMRP expression in animal models. Sensorimotor gating as measured by pre-pulse inhibition (PPI) is altered in both FXS patients and Fmr1 knock out (KO) mice. Similarly, FXTAS patients have demonstrated PPI deficits. Recent work suggests there may be overlapping synaptic defects between Fmr1 KO and CGG knock-in premutation mouse models (CGG KI). We therefore sought to interrogate PPI in CGG KI mice. Using a quiet PPI protocol more akin to human testing conditions, we find that Fmr1 KO animals have significantly impaired PPI. Using this same protocol, we find CGG KI mice demonstrate an age-dependent impairment in PPI compared to wild type (WT) controls. This study describes a novel phenotype in CGG KI mice that can be used in future therapeutic development targeting premutation associated symptoms.

Sensory stimulation increases cortical expression of the fragile X mental retardation protein in vivo.

Fragile X syndrome is a common cause of mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), an RNA binding protein whose function remains unclear. Recent in vitro work has demonstrated that the protein is translated near the synapse in an activity dependent manner [33]. We therefore asked whether expression of FMRP might be altered by neuronal activity in vivo. Using immunoblots of different sub-cellular fractions of the rat somatosensory cortex, we show that the levels of FMRP increase significantly following unilateral whisker stimulation, a model of experience dependent plasticity. This increase is greatest between 2 and 8 h after the stimulus and is seen in both a synaptosomal fraction as well as a sub-cellular fraction enriched for polyribosomal complexes. In contrast, detectable levels of FMRP within the somatosensory cortex show either a decrease or no change after a kainic acid induced seizure compared to water treated controls. Our findings demonstrate that FMRP expression levels are modulated in vivo in response to neuronal activity and suggest a role for FMRP in activity dependent plasticity.

Behavioral sensitization and extracellular dopamine responses to amphetamine after various treatments.

The repeated administration of amphetamine (AMPH) results in a pattern of behavioral changes which includes an augmentation of some behaviors, generally referred to as behavioral sensitization. Some investigators have suggested that an increased dopamine (DA) response to AMPH challenge may underlie behavioral sensitization, while others have reported behavioral sensitization in the absence of an enhanced DA response. Because temporal and dosage parameters of the AMPH pretreatment regimen have been suggested to play a role in the appearance of an enhanced DA response, we utilized a variety of AMPH pretreatment regimens to assess the relationship between pretreatment dose of AMPH, duration of withdrawal and the DA response in caudate-putamen and nucleus accumbens to a subsequent AMPH challenge. Under our experimental conditions, behavioral sensitization was observed after each of these treatments in the absence of an enhanced DA response in either brain region.