Fragile X mental retardation protein (FMRP), an RNA binding protein (RBP), is aberrantly hyper-expressed in human tumors and plays an essential role in tumor invasion, metastasis and immune evasion. However, there is no small-molecule inhibitor for FMRP so far. In this study, we developed the first FMRP-targeting degrader based on PROteolysis TArgeting Chimera (PROTAC) technology and constructed a heterobifunctional PROTAC through linking a FMRP-targeting G-quadruplex RNA (sc1) to a von Hippel-Lindau (VHL)-targeting ligand peptide (named as sc1-VHLL). Sc1-VHLL specifically degraded endogenous FMRP via ubiquitination pathway in both mouse and human cancer cells. The FMRP degradation significantly changed the secretion pattern of cancer cells, resulting in higher expression of pro-inflammatory cytokine and smaller amounts of immunomodulatory contents. Furthermore, sc1-VHLL, when encapsulated into ionizable liposome nanoparticles (LNP) efficiently targeted tumor site and degraded FMRP in cancer cells. In CT26 tumor-bearing mouse model, FMRP degradation within tumors substantially promoted the infiltration of lymphocytes and CD8 T cells and reduced the proportion of Treg cells, reshaping the proinflammatory tumor microenvironment and accordingly transforming cold tumor into hot tumor. When combined with immune checkpoint blockade (ICB) therapy, sc1-VHLL based treatment remarkably inhibited the tumor growth.

Fragile X syndrome (FXS) is caused by the full mutation in the FMR1 gene on the Xq27.3 chromosome region. It is the most common monogenic cause of autism spectrum disorder (ASD) and inherited intellectual disability (ID). Besides ASD and ID and other symptoms, individuals with FXS may exhibit sleep problems and impairment of circadian rhythm (CR). The Drosophila melanogaster models of FXS, such as dFMR1B55, represent excellent models for research in the FXS field. During this study, sleep patterns and CR in dFMR1B55 mutants were analyzed, using a new platform based on continuous high-resolution videography integrated with a highly-customized version of an open-source software. This methodology provides more sensitive results, which could be crucial for all further research in this model of fruit flies. The study revealed that dFMR1B55 male mutants sleep more and can be considered weak rhythmic flies rather than totally arrhythmic and present a good alternative animal model of genetic disorder, which includes impairment of CR and sleep behavior. The combination of affordable videography and software used in the current study is a significant improvement over previous methods and will enable broader adaptation of such high-resolution behavior monitoring methods.

Fragile X syndrome (FXS) is the most common genetic cause of autism spectrum disorder engendered by transcriptional silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene. Given the early onset of behavioral and molecular changes, it is imperative to know the optimal timing for therapeutic intervention. Case reports documented benefits of metformin treatment in FXS children between 2 and 14 y old. In this study, we administered metformin from birth to Fmr1-/y mice which corrected up-regulated mitogen-2 activated protein kinase/extracellular signal-regulated kinase and mammalian/mechanistic target of rapamycin complex 1 signaling pathways and specific synaptic mRNA-binding targets of FMRP. Metformin rescued increased number of calls in ultrasonic vocalization and repetitive behavior in Fmr1-/y mice. Our findings demonstrate that in mice, early-in-life metformin intervention is effective in treating FXS pathophysiology.

Behavioral and pharmaceutical interventions reverse defects associated with increased cerebellar long-term depression in a mouse model of Fragile X syndrome.

Prostate adenocarcinoma (PRAD) is the second most common tumor associated with death. The role and mechanisms of the fragile X mental retardation 1 (FMR1) gene in PRAD remain unknown. We conducted an analysis of FMR1 expression in PRAD to determine its prognostic importance and connection to carcinogenic pathways such as PI3K_AKT_mTOR. Survival analyses were utilized to establish a correlation between FMR1 expression and patient outcomes. We used the integration of genomic data with bioinformatic predictions to predict the regulatory factors of the FMR1 gene in PRAD. Our data revealed that individuals with higher levels of FMR1 expression experience worse survival outcomes compared to those with lower expression (hazard ratio [HR] = 5.08, 95% confidence interval [CI] = 1.07 - 24, p = 0.0412). FMR1 expression was significantly higher in patients with advanced pathological tumor stages, particularly in the pT3 and pT4 combined stages and the pN1 nodal stage. Furthermore, patients with high Gleason scores (GSs) (combined GSs 8 and 9) exhibited increased levels of FMR1 expression. Our results further identify a possible regulatory link between FMR1 and key oncogenic pathways, including PI3K_AKT_mTOR, and predict the possible mechanism by which FMR1 is regulated in PRAD. Our data suggest that the FMR1 gene could serve as a biomarker for PRAD progression. However, in-depth investigations, including those with large patient samples and in vitro studies, are needed to validate this finding and understand the mechanisms involved.

Fragile X syndrome (FXS) is an intellectual developmental disorder characterized, inter alia, by deficits in the short-term processing of neural information, such as sensory processing and working memory. The primary cause of FXS is the loss of fragile X messenger ribonucleoprotein (FMRP), which is profoundly involved in synaptic function and plasticity. Short-term synaptic plasticity (STSP) may play important roles in functions that are affected by FXS. Recent evidence points to the crucial involvement of the presynaptic calcium sensor synaptotagmin-7 (Syt-7) in STSP. However, how the loss of FMRP affects STSP and Syt-7 have been insufficiently studied. Furthermore, males and females are affected differently by FXS, but the underlying mechanisms remain elusive. The aim of the present study was to investigate possible changes in STSP and the expression of Syt-7 in the dorsal (DH) and ventral (VH) hippocampus of adult males and females in a Fmr1-knockout (KO) rat model of FXS. We found that the paired-pulse ratio (PPR) and frequency facilitation/depression (FF/D), two forms of STSP, as well as the expression of Syt-7, are normal in adult KO males, but the PPR is increased in the ventral hippocampus of KO females (6.4 ± 3.7 vs. 18.3 ± 4.2 at 25 ms in wild type (WT) and KO, respectively). Furthermore, we found no gender-related differences, but did find robust region-dependent difference in the STSP (e.g., the PPR at 50 ms: 50.0 ± 5.5 vs. 17.6 ± 2.9 in DH and VH of WT male rats; 53.1 ± 3.6 vs. 19.3 ± 4.6 in DH and VH of WT female rats; 48.1 ± 2.3 vs. 19.1 ± 3.3 in DH and VH of KO male rats; and 51.2 ± 3.3 vs. 24.7 ± 4.3 in DH and VH of KO female rats). AMPA receptors are similarly expressed in the two hippocampal segments of the two genotypes and in both genders. Also, basal excitatory synaptic transmission is higher in males compared to females. Interestingly, we found more than a twofold higher level of Syt-7, not synaptotagmin-1, in the dorsal compared to the ventral hippocampus in the males of both genotypes (0.43 ± 0.1 vs. 0.16 ± 0.02 in DH and VH of WT male rats, and 0.6 ± 0.13 vs. 0.23 ± 0.04 in DH and VH of KO male rats) and in the WT females (0.97 ± 0.23 vs. 0.31 ± 0.09 in DH and VH). These results point to the susceptibility of the female ventral hippocampus to FMRP loss. Importantly, the different levels of Syt-7, which parallel the higher score of the dorsal vs. ventral hippocampus on synaptic facilitation, suggest that Syt-7 may play a pivotal role in defining the striking differences in STSP along the long axis of the hippocampus.

OBJECTIVE: Fragile X Syndrome (FXS) is the most common cause of inherited intellectual disability, caused by CGG-repeat expansions (> 200) in the FMR1 gene leading to lack of expression. Espansion between 55 and 200 triplets fall within the premutation range (PM) and can lead to different clinical conditions, including fragile X- primary ovarian insufficiency (FXPOI), fragile X-associated neuropsychiatric disorders (FXAND) and fragile X-associated tremor/ataxia syndrome (FXTAS). Although there is not a current cure for FXS and for the Fragile X-PM associated conditions (FXPAC), timely diagnosis as well as the implementation of treatment strategies, psychoeducation and behavioral intervention may improve the quality of life (QoL) of people with FXS or FXPAC. With the aim to investigate the main areas of concerns and the priorities of treatment in these populations, the Italian National Fragile X Association in collaboration with Bambino Gesù Children\'s Hospital, conducted a survey among Italian participants.
METHODS: Here, we present a survey based on the previous study that Weber and colleagues conducted in 2019 and that aimed to investigate the main symptoms and challenges in American individuals with FXS. The survey has been translated into Italian language to explore FXS needs of treatment also among Italian individuals affected by FXS, family members, caretakers, and professionals. Furthermore, we added a section designated only to people with PM, to investigate the main symptoms, daily living challenges and treatment priorities.
RESULTS: Anxiety, challenging behaviors, language difficulties and learning disabilities were considered the major areas of concern in FXS, while PM was reported as strongly associated to cognitive problems, social anxiety, and overthinking. Anxiety was reported as a treatment priority in both FXS and PM.
CONCLUSIONS: FXS and PM can be associated with a range of cognitive, affective, and physical health complications. Taking a patient-first perspective may help clinicians to better characterize the cognitive-behavioral phenotype associated to these conditions, and eventually to implement tailored therapeutic approaches.

Fragile X syndrome (FXS), the most frequent monogenic form of intellectual disability, is caused by transcriptional silencing of the FMR1 gene that could render neuronal hyperexcitability. Here we show that pyramidal cells (PCs) in the dorsal CA1 region of the hippocampus elicited a larger action potential (AP) number in response to suprathreshold stimulation in juvenile Fmr1 knockout (KO) than wild-type (WT) mice. Because Kv7/M channels modulate CA1 PC excitability in rats, we investigated if their dysfunction produces neuronal hyperexcitability in Fmr1 KO mice. Immunohistochemical and western blot analyses showed no differences in the expression of Kv7.2 and Kv7.3 channel subunits between genotypes; however, the current mediated by Kv7/M channels was reduced in Fmr1 KO mice. In both genotypes, bath application of XE991 (10 μM), a blocker of Kv7/M channels: produced an increased AP number, produced an increased input resistance, produced a decreased AP voltage threshold and shaped AP medium afterhyperpolarization by increasing mean velocities. Retigabine (10 μM), an opener of Kv7/M channels, produced opposite effects to XE991. Both XE991 and retigabine abolished differences in all these parameters found in control conditions between genotypes. Furthermore, a low concentration of retigabine (2.5 μM) normalized CA1 PC excitability of Fmr1 KO mice. Finally, ex vivo seizure-like events evoked by 4-aminopyiridine (200 μM) in the dorsal CA1 region were more frequent in Fmr1 KO mice, and were abolished by retigabine (5-10 μM). We conclude that CA1 PCs of Fmr1 KO mice exhibit hyperexcitability, caused by Kv7/M channel dysfunction, and increased epileptiform activity, which were abolished by retigabine. KEY POINTS: Dorsal pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice exhibit hyperexcitability. Kv7/M channel activity, but not expression, is reduced in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Kv7/M channel dysfunction causes hyperexcitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice by increasing input resistance, decreasing AP voltage threshold and shaping medium afterhyperpolarization. A Kv7/M channel opener normalizes neuronal excitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Ex vivo seizure-like events evoked in the dorsal CA1 region were more frequent in Fmr1 KO mice, and such an epileptiform activity was abolished by a Kv7/M channel opener depending on drug concentration. Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome.

Although hyperactivity is associated with a wide variety of neurodevelopmental disorders, the early embryonic origins of locomotion have hindered investigation of pathogenesis of these debilitating behaviors. The earliest motor output in vertebrate animals is generated by clusters of early-born motor neurons (MNs) that occupy distinct regions of the spinal cord, innervating stereotyped muscle groups. Gap junction electrical synapses drive early spontaneous behavior in zebrafish, prior to the emergence of chemical neurotransmitter networks. We use a genetic model of hyperactivity to gain critical insight into the consequences of errors in motor circuit formation and function, finding that Fragile X syndrome model mutant zebrafish are hyperexcitable from the earliest phases of spontaneous behavior, show altered sensitivity to blockade of electrical gap junctions, and have increased expression of the gap junction protein Connexin 34/35. We further show that this hyperexcitable behavior can be rescued by pharmacological inhibition of electrical synapses. We also use functional imaging to examine MN and interneuron (IN) activity in early embryogenesis, finding genetic disruption of electrical gap junctions uncouples activity between mnx1 + MNs and INs. Taken together, our work highlights the importance of electrical synapses in motor development and suggests that the origins of hyperactivity in neurodevelopmental disorders may be established during the initial formation of locomotive circuits.

The enhancement of associative synaptic plasticity often results in impaired rather than enhanced learning. Previously, we proposed that such learning impairments can result from saturation of the plasticity mechanism (Nguyen-Vu et al., 2017), or, more generally, from a history-dependent change in the threshold for plasticity. This hypothesis was based on experimental results from mice lacking two class I major histocompatibility molecules, MHCI H2-Kb and H2-Db (MHCI KbDb-/-), which have enhanced associative long-term depression at the parallel fiber-Purkinje cell synapses in the cerebellum (PF-Purkinje cell LTD). Here, we extend this work by testing predictions of the threshold metaplasticity hypothesis in a second mouse line with enhanced PF-Purkinje cell LTD, the Fmr1 knockout mouse model of Fragile X syndrome (FXS). Mice lacking Fmr1 gene expression in cerebellar Purkinje cells (L7-Fmr1 KO) were selectively impaired on two oculomotor learning tasks in which PF-Purkinje cell LTD has been implicated, with no impairment on LTD-independent oculomotor learning tasks. Consistent with the threshold metaplasticity hypothesis, behavioral pre-training designed to reverse LTD at the PF-Purkinje cell synapses eliminated the oculomotor learning deficit in the L7-Fmr1 KO mice, as previously reported in MHCI KbDb-/-mice. In addition, diazepam treatment to suppress neural activity and thereby limit the induction of associative LTD during the pre-training period also eliminated the learning deficits in L7-Fmr1 KO mice. These results support the hypothesis that cerebellar LTD-dependent learning is governed by an experience-dependent sliding threshold for plasticity. An increased threshold for LTD in response to elevated neural activity would tend to oppose firing rate stability, but could serve to stabilize synaptic weights and recently acquired memories. The metaplasticity perspective could inform the development of new clinical approaches for addressing learning impairments in autism and other disorders of the nervous system.