Fragile X-associated tremor/ataxia syndrome (FXTAS) is a rare neurodegenerative disorder caused by a 55-200 CGG repeat expansion in the 5' untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. FXTAS is characterized by progressive cerebellar ataxia, Parkinsonism, intention tremors and cognitive decline. The main neuropathological hallmark of FXTAS is the presence of ubiquitin-positive intranuclear inclusions in neurons and astrocytes throughout the brain. The molecular pathology of FXTAS involves the presence of 2 to 8-fold elevated levels of FMR1 mRNA, and of a repeat-associated non-AUG (RAN) translated polyglycine peptide (FMRpolyG). Increased levels of FMR1 mRNA containing an expanded CGG repeat can result in cellular toxicity by an RNA gain-of-function mechanism. The increased levels of CGG repeat-expanded FMR1 transcripts may create RNA foci that sequester important cellular proteins, including RNA-binding proteins and FMRpolyG, in intranuclear inclusions. To date, it is unclear whether the FMRpolyG-positive intranuclear inclusions are a cause or a consequence of FXTAS disease pathology. In this report we studied the relation between the presence of neuronal intranuclear inclusions and behavioral deficits using an inducible mouse model for FXTAS. Neuronal intranuclear inclusions were observed 4 weeks after dox-induction. After 12 weeks, high numbers of FMRpolyG-positive intranuclear inclusions could be detected in the hippocampus and striatum, but no clear signs of behavioral deficits related to these specific brain regions were found. In conclusion, the observations in our inducible mouse model for FXTAS suggest a lack of correlation between the presence of intranuclear FMRpolyG-positive aggregates in brain regions and specific behavioral phenotypes.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an incurable neurodegenerative disorder caused by expansion of CGG repeats in the FMR1 5'UTR. The RNA containing expanded CGG repeats (rCGGexp) causes cell damage by interaction with complementary DNA, forming R-loop structures, sequestration of nuclear proteins involved in RNA metabolism and initiation of translation of polyglycine-containing protein (FMRpolyG), which forms nuclear insoluble inclusions. Here we show the therapeutic potential of short antisense oligonucleotide steric blockers (ASOs) targeting directly the rCGGexp. In nuclei of FXTAS cells ASOs affect R-loop formation and correct miRNA biogenesis and alternative splicing, indicating that nuclear proteins are released from toxic sequestration. In cytoplasm, ASOs significantly decrease the biosynthesis and accumulation of FMRpolyG. Delivery of ASO into a brain of FXTAS mouse model reduces formation of inclusions, improves motor behavior and corrects gene expression profile with marginal signs of toxicity after a few weeks from a treatment.

CGG repeat expansions within the premutation range (55-200) of the FMR1 gene can lead to Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders. These CGG repeats are translated into a toxic polyglycine-containing protein, FMRpolyG. Pathology of Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders comprises FMRpolyG- and p62-positive intranuclear inclusions. Diagnosing a FMR1-premutation carrier remains challenging, as the clinical features overlap with other neurodegenerative diseases. Here, we describe two male cases with Fragile X-associated neuropsychiatric disorders-related symptoms and mild movement disturbances and novel pathological features that can attribute to the variable phenotype. Macroscopically, both donors did not show characteristic white matter lesions on MRI; however, vascular infarcts in cortical- and sub-cortical regions were identified. Immunohistochemistry analyses revealed a high number of FMRpolyG intranuclear inclusions throughout the brain, which were also positive for p62. Importantly, we identified a novel pathological vascular phenotype with inclusions present in pericytes and endothelial cells. Although these results need to be confirmed in more cases, we propose that these vascular lesions in the brain could contribute to the complex symptomology of FMR1-premutation carriers. Overall, our report suggests that Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders may present diverse clinical involvements resembling other types of dementia, and in the absence of genetic testing, FMRpolyG can be used post-mortem to identify premutation carriers.

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a late onset, recessively inherited neurodegenerative disorder caused by biallelic, non-reference pentameric AAGGG(CCCTT) repeat expansions within the second intron of replication factor complex subunit 1 (RFC1). To investigate how these repeats cause disease, we generated CANVAS patient induced pluripotent stem cell (iPSC) derived neurons (iNeurons) and utilized calcium imaging and transcriptomic analysis to define repeat-elicited gain-of-function and loss-of-function contributions to neuronal toxicity. AAGGG repeat expansions do not alter neuronal RFC1 splicing, expression, or DNA repair pathway functions. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins that selectively accumulate in CANVAS patient brains. However, neither these proteins nor repeat RNA foci were detected in iNeurons, and overexpression of these repeats in isolation did not induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded allele. These phenotypic deficits were not replicated by knockdown of RFC1 in control neurons and were not rescued by ectopic expression of RFC1. These findings support a repeat-dependent but RFC1-independent cause of neuronal dysfunction in CANVAS, with important implications for therapeutic development in this currently untreatable condition.

Recent evidence indicates that specific RNAs promote the formation of ribonucleoprotein condensates by acting as scaffolds for RNA-binding proteins (RBPs). We systematically investigated RNA-RBP interaction networks to understand ribonucleoprotein assembly. We found that highly contacted RNAs are structured, have long UTRs, and contain nucleotide repeat expansions. Among the RNAs with such properties, we identified the FMR1 3' UTR that harbors CGG expansions implicated in fragile X-associated tremor/ataxia syndrome (FXTAS). We studied FMR1 binding partners in silico and in vitro and prioritized the splicing regulator TRA2A for further characterization. In a FXTAS cellular model, we validated the TRA2A-FMR1 interaction and investigated implications of its sequestration at both transcriptomic and post-transcriptomic levels. We found that TRA2A co-aggregates with FMR1 in a FXTAS mouse model and in post-mortem human samples. Our integrative study identifies key components of ribonucleoprotein aggregates, providing links to neurodegenerative disease and allowing the discovery of therapeutic targets.

Fragile X syndrome (FXS) is a common monogenetic cause of intellectual disability, autism spectrum features, and a broad range of other psychiatric and medical problems. FXS is caused by the lack of the fragile X mental retardation protein (FMRP), a translational regulator of specific mRNAs at the postsynaptic compartment. The absence of FMRP leads to aberrant synaptic plasticity, which is believed to be caused by an imbalance in excitatory and inhibitory network functioning of the synapse. Evidence from studies in mice demonstrates that GABA, the major inhibitory neurotransmitter in the brain, and its receptors, is involved in the pathogenesis of FXS. Moreover, several FXS phenotypes, including social behavior deficits, could be corrected in Fmr1 KO mice after acute treatment with GABAB agonists.

Although the right temporal variant frontotemporal dementia (rtvFTD) is characterised by distinct clinical and radiological features, its underlying histopathology remains elusive. Being considered a right-sided variant of semantic variant primary progressive aphasia (svPPA), TDP-43 type C pathology has been linked to the syndrome, but this has not been studied in detail in large cohorts. In this case report and systematic review, we report the autopsy results of five subjects diagnosed with rtvFTD from our cohort and 44 single rtvFTD subjects from the literature. Macroscopic pathological evaluation of the combined results revealed that rtvFTD demonstrated either a frontotemporal or temporal evolution, even if the degeneration started in the right temporal lobe initially. FTLD-TDP type C was the most common underlying pathology in rtvFTD, however, in 64% of rtvFTD, other underlying pathologies than FTLD-TDP type C were present, such as Tau-MAPT and FTLD-TDP type A and B. Additionally, accompanying motor neuron or corticospinal tract degeneration was observed in 28% of rtvFTD patients. Our results show that in contrast to the general assumption, rtvFTD might not be a pure FTLD-TDP type C disorder, unlike its left temporal counterpart svPPA. Large sample size pathological studies are warranted to understand the diverse pathologies of the right and left temporal variants of frontotemporal dementia.

Fragile X premutation disorder is caused by CGG triplet repeat expansions in the 5' untranslated region of FMR1 mRNA. The question of how expanded CGG repeats cause disease is a subject of continuing debate. Our work indicates that CGG-repeat structures compete with regulatory BC1 RNA for access to RNA transport factor hnRNP A2. As a result, BC1 RNA is mislocalized in vivo, as its synapto-dendritic presence is severely diminished in brains of CGG-repeat knock-in animals (a premutation mouse model). Lack of BC1 RNA is known to cause seizure activity and cognitive dysfunction. Our working hypothesis thus predicted that absence, or significantly reduced presence, of BC1 RNA in synapto-dendritic domains of premutation animal neurons would engender cognate phenotypic alterations. Testing this prediction, we established epileptogenic susceptibility and cognitive impairments as major phenotypic abnormalities of CGG premutation mice. In CA3 hippocampal neurons of such animals, synaptic release of glutamate elicits neuronal hyperexcitability in the form of group I metabotropic glutamate receptor-dependent prolonged epileptiform discharges. CGG-repeat knock-in animals are susceptible to sound-induced seizures and are cognitively impaired as revealed in the Attentional Set Shift Task. These phenotypic disturbances occur in young-adult premutation animals, indicating that a neurodevelopmental deficit is an early-initial manifestation of the disorder. The data are consistent with the notion that RNA mislocalization can contribute to pathogenesis.

The fragile X premutation is a CGG trinucleotide repeat expansion between 55 and 200 repeats in the 5'-untranslated region of the fragile X mental retardation 1 (FMR1) gene. Human carriers of the premutation allele are at risk of developing the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Characteristic neuropathology associated with FXTAS includes intranuclear inclusions in neurons and astroglia. Previous studies recapitulated these histopathological features in neurons in a knock-in mouse model, but without significant astroglial pathology. To determine the role of astroglia in FXTAS, we generated a transgenic mouse line (Gfa2-CGG99-eGFP) that selectively expresses a 99-CGG repeat expansion linked to an enhanced green fluorescent protein (eGFP) reporter in astroglia throughout the brain, including cerebellar Bergmann glia. Behaviorally these mice displayed impaired motor performance on the ladder-rung test, but paradoxically better performance on the rotarod. Immunocytochemical analysis revealed that CGG99-eGFP co-localized with GFAP and S-100ß, but not with NeuN, Iba1, or MBP, indicating that CGG99-eGFP expression is specific to astroglia. Ubiquitin-positive intranuclear inclusions were found in eGFP-expressing glia throughout the brain. In addition, intracytoplasmic ubiquitin-positive inclusions were found outside the nucleus in distal astrocyte processes. Intriguingly, intranuclear inclusions, in the absence of eGFP mRNA and eGFP fluorescence, were present in neurons of the hypothalamus and neocortex. Furthermore, intranuclear inclusions in both neurons and astrocytes displayed immunofluorescent labeling for the polyglycine peptide FMRpolyG, implicating FMRpolyG in the pathology found in Gfa2-CGG99 mice. Considered together, these results show that Gfa2-CGG99 expression in mice is sufficient to induce key features of FXTAS pathology, including formation of intranuclear inclusions, translation of FMRpolyG, and deficits in motor function.

Fragile X-associated tremor ataxia syndrome (FXTAS) is a rare disorder associated to the presence of the fragile X premutation, a 55-200 CGG repeat expansion in the 5' UTR of the FMR1 gene. Two main neurological phenotypes have been described in carriers of the CGG premutation: (1) neurodevelopmental disorders characterized by anxiety, attention deficit hyperactivity disorder (ADHD), social deficits, or autism spectrum disorder (ASD); and (2) after 50 years old, the FXTAS phenotype. This neurodegenerative disorder is characterized by ataxia and a form of parkinsonism. The molecular pathology of this disorder is characterized by the presence of elevated levels of Fragile X Mental Retardation 1 (FMR1) mRNA, presence of a repeat-associated non-AUG (RAN) translated peptide, and FMR1 mRNA-containing nuclear inclusions. Whereas in the past FXTAS was mainly considered as a late-onset disorder, some phenotypes of patients and altered learning and memory behavior of a mouse model of FXTAS suggested that this disorder involves neurodevelopment. To better understand the physiopathological role of the increased levels of Fmr1 mRNA during neuronal differentiation, we used a small interfering RNA (siRNA) approach to reduce the abundance of this mRNA in cultured cortical neurons from the FXTAS mouse model. Morphological alterations of neurons were rescued by this approach. This cellular phenotype is associated to differentially expressed proteins that we identified by mass spectrometry analysis. Interestingly, phenotype rescue is also associated to the rescue of the abundance of 29 proteins that are involved in various pathways, which represent putative targets for early therapeutic approaches.

Frontotemporal Dementia (FTD) is caused by frontal-temporal lobar degeneration (FTLD), characterized mainly by brain protein aggregates of tau (FTLD-Tau) or TDP-43 (FTLD-TDP). The clinicopathological heterogeneity makes ante-mortem diagnosis of these pathological subtypes challenging. Our proteomics study showed increased Apolipoprotein L1 (APOL1) levels in CSF from FTD patients, which was prominently expressed in FTLD-Tau. We aimed to understand APOL1 expression in FTLD post-mortem brain tissue and to validate its potential as a CSF biomarker for FTD and its pathological subtypes.

Many RNA-binding proteins (RBPs), particularly those associated with RNA granules, promote pathological protein aggregation in neurodegenerative diseases. Here, we demonstrate that G3BP2, a core component of stress granules, directly interacts with Tau and inhibits Tau aggregation. In the human brain, the interaction of G3BP2 and Tau is dramatically increased in multiple tauopathies, and it is independent of neurofibrillary tangle (NFT) formation in Alzheimer's disease (AD). Surprisingly, Tau pathology is significantly elevated upon loss of G3BP2 in human neurons and brain organoids. Moreover, we found that G3BP2 masks the microtubule-binding region (MTBR) of Tau, thereby inhibiting Tau aggregation. Our study defines a novel role for RBPs as a line of defense against Tau aggregation in tauopathies.

The clinical phenotype of the rare behavioural variant of Alzheimer's disease (bvAD) is insufficiently understood. Given the strong clinico-anatomical correlations of tau pathology in AD, we investigated the distribution of tau deposits in bvAD, in-vivo and ex-vivo, using positron emission tomography (PET) and postmortem examination.

Subjects with incidental Lewy body disease (iLBD) may represent the premotor stage of Parkinson's disease (PD). To elucidate molecular mechanisms underlying neuronal dysfunction and alpha-synuclein pathology in the premotor phase of PD, we investigated the transcriptome of the substantia nigra (SN) of well-characterized iLBD, PD donors and age-matched controls with Braak alpha-synuclein stage ranging from 0-6. In Braak alpha-synuclein stages 1 and 2, we observed deregulation of pathways linked to axonal degeneration, immune response and endocytosis, including axonal guidance signaling, mTOR signaling, EIF2 signaling and clathrin-mediated endocytosis in the SN. In Braak stages 3 and 4, we observed deregulation of pathways involved in protein translation and cell survival, including mTOR and EIF2 signaling. In Braak stages 5 and 6, we observed deregulation of dopaminergic signaling, axonal guidance signaling and thrombin signaling. Throughout the progression of PD pathology, we observed a deregulation of mTOR, EIF2 and regulation of eIF4 and p70S6K signaling in the SN. Our results indicate that molecular mechanisms related to axonal dysfunction, endocytosis and immune response are an early event in PD pathology, whereas mTOR and EIF2 signaling are impaired throughout disease progression. These pathways may hold the key to altering the disease progression in PD.

Fragile X-associated tremor and ataxia syndrome (FXTAS) is a late-onset, progressive neurodegenerative disorder characterized by tremors, ataxia and neuropsychological problems. This disease is quite common in the general population with approximately 20 million carriers worldwide. The risk of developing FXTAS increases dramatically with age, with about 45% of male carriers over the age of 50 being affected. FXTAS is caused by a CGG-repeat expansion (CGGexp) in the fragile X mental retardation 1 (FMR1) gene. CGGexp RNA is translated into the FMRpolyG protein by a mechanism called RAN translation. Although both gene and pathogenic trigger are known, no therapeutic interventions are available at this moment. Here, we present, for the first time, primary hippocampal neurons derived from the ubiquitous inducible mouse model which is used as a screening tool for targeted interventions. A promising candidate is the repeat binding, RAN translation blocking, small molecule 1a. Small molecule 1a shields the disease-causing CGGexp from being translated into the toxic FMRpolyG protein. Primary hippocampal neurons formed FMRpolyG-positive inclusions, and upon treatment with 1a, the numbers of FMRpolyG-positive inclusions are reduced. We also describe for the first time the formation of FMRpolyG-positive inclusions in the liver of this mouse model. Treatment with 1a reduced the insoluble FMRpolyG protein fraction in the liver but not the number of inclusions. Moreover, 1a treatment had a reducing effect on the number of Rad23b-positive inclusions and insoluble Rad23b protein levels. These data suggest that targeted small molecule therapy is effective in an FXTAS mouse model and has the potential to treat CGGexp-mediated diseases, including FXTAS.

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease characterized by the presence of intranuclear inclusions of unknown origin. NIID is caused by an expansion of GGC repeats in the 5' UTR of the NOTCH2NLC (N2C) gene. We found that these repeats are embedded in a small upstream open reading frame (uORF) (uN2C), resulting in their translation into a polyglycine-containing protein, uN2CpolyG. This protein accumulates in intranuclear inclusions in cell and mouse models and in tissue samples of individuals with NIID. Furthermore, expression of uN2CpolyG in mice leads to locomotor alterations, neuronal cell loss, and premature death of the animals. These results suggest that translation of expanded GGC repeats into a novel and pathogenic polyglycine-containing protein underlies the presence of intranuclear inclusions and neurodegeneration in NIID.

A repeat expansion in the C9orf72 gene is the most prevalent genetic cause of frontotemporal dementia (C9-FTD). Several studies have indicated the involvement of the unfolded protein response (UPR) in C9-FTD. In human neuropathology, UPR markers are strongly associated with granulovacuolar degeneration (GVD). In this study, we aim to assess the presence of UPR markers together with the presence of dipeptide pathology and GVD in post mortem brain tissue from C9-FTD cases and neurologically healthy controls. Using immunohistochemistry we assessed the presence of phosphorylated PERK, IRE1α and eIF2α in the frontal cortex, hippocampus and cerebellum of C9-FTD (n = 18) and control (n = 9) cases. The presence of UPR activation markers was compared with the occurrence of pTDP-43, p62 and dipeptide repeat (DPR) proteins (poly(GA), -(GR) & -(GP)) as well as casein kinase 1 delta (CK1δ), a marker for GVD. Increased presence of UPR markers was observed in the hippocampus and cerebellum in C9-FTD compared to control cases. In the hippocampus, overall levels of pPERK and peIF2α were higher in C9-FTD, including in granule cells of the dentate gyrus (DG). UPR markers were also observed in granule cells of the cerebellum in C9-FTD. In addition, increased levels of CK1δ were observed in granule cells in the DG of the hippocampus and granular layer of the cerebellum in C9-FTD. Double-labelling experiments indicate a strong association between UPR markers and the presence of dipeptide pathology as well as GVD. We conclude that UPR markers are increased in C9-FTD and that their presence is associated with dipeptide pathology and GVD. Increased presence of UPR markers and CK1δ in granule cells in the cerebellum and hippocampus could be a unique feature of C9-FTD.

Movement disorders (MD) have been linked to degeneration of the substantia nigra (SN) in Parkinson's disease and include bradykinesia, rigidity, and tremor. They are also present in frontotemporal dementia (FTD), where MD have been linked to frontotemporal lobar degeneration with tau pathology (FTLD-tau). Although MD can also occur in FTLD with TDP-43 pathology (FTLD-TDP), the local pathology in the SN of FTLD-TDP patients with MD is currently unexplored. The aims of this study are to characterize the frequency and the nature of MD in a cohort of FTLD-TDP brain donors and to investigate the relationship between the presence of MD, the nigral neuronal loss, and the TDP-43 burden in the SN. From our cohort of FTLD-TDP patients (n = 53), we included 13 donors who presented with MD (FTLD-MD+), and nine age-sex matched donors without MD (FTLD-MD-) for whom the SN was available. In these donors, the TDP-43 burden and the neuronal density in the SN were assessed with ImageJ and Qupath software. The results were compared between the two groups using T-test. We found that the TDP-43 burden in the SN was higher in FTLD-MD+ (mean 3,43%, SD ± 2,7) compared to FTLD-MD- (mean 1,21%, SD ± 0,67) (p = 0,04), while no significant difference in nigral neuronal density was found between the groups (p = 0,09). 17% of FTLD-TDP patients developed MD, which present as symmetric akinetic-rigid parkinsonism or CBS. Given the absence of a significant nigral neuronal cell loss, TDP-43 induced neuronal dysfunction could be sufficient to cause MD.