Tissue-specific and repeat length-dependent somatic instability of the X-linked dystonia parkinsonism-associated CCCTCT repeat.

X-linked dystonia-parkinsonism (XDP) is a progressive adult-onset neurodegenerative disorder caused by insertion of a SINE-VNTR-Alu (SVA) retrotransposon in the TAF1 gene. The SVA retrotransposon contains a CCCTCT hexameric repeat tract of variable length, whose length is inversely correlated with age at onset. This places XDP in a broader class of repeat expansion diseases, characterized by the instability of their causative repeat mutations. Here, we observe similar inverse correlations between CCCTCT repeat length with age at onset and age at death and no obvious correlation with disease duration. To gain insight into repeat instability in XDP we performed comprehensive quantitative analyses of somatic instability of the XDP CCCTCT repeat in blood and in seventeen brain regions from affected males. Our findings reveal repeat length-dependent and expansion-based instability of the XDP CCCTCT repeat, with greater levels of expansion in brain than in blood. The brain exhibits regional-specific patterns of instability that are broadly similar across individuals, with cerebellum exhibiting low instability and cortical regions exhibiting relatively high instability. The spectrum of somatic instability in the brain includes a high proportion of moderate repeat length changes of up to 5 repeats, as well as expansions of ~ 20- > 100 repeats and contractions of ~ 20-40 repeats at lower frequencies. Comparison with HTT CAG repeat instability in postmortem Huntington's disease brains reveals similar brain region-specific profiles, indicating common trans-acting factors that contribute to the instability of both repeats. Analyses in XDP brains of expansion of a different SVA-associated CCCTCT located in the LIPG gene, and not known to be disease-associated, reveals repeat length-dependent expansion at overall lower levels relative to the XDP CCCTCT repeat, suggesting that expansion propensity may be modified by local chromatin structure. Together, the data support a role for repeat length-dependent somatic expansion in the process(es) driving the onset of XDP and prompt further investigation into repeat dynamics and the relationship to disease.

Dissection of TAF1 neuronal splicing and implications for neurodegeneration in X-linked dystonia-parkinsonism.

X-linked dystonia-parkinsonism (XDP) is a monogenic neurodegenerative disorder of the basal ganglia, which presents as a combination of hyperkinetic movements and parkinsonian features. The underlying genetic mechanism involves the insertion of a SINE-VNTR-Alu retrotransposon within the TAF1 gene. Interestingly, alterations of TAF1 have been involved in multiple neurological diseases. In XDP, the SINE-VNTR-Alu insertion in TAF1 has been proposed to result in alternative splicing defects, including the decreased incorporation of a neuron-specific microexon annotated as 34'. This mechanism has become controversial as recent studies failed to provide support. In order to resolve this conundrum, we examined the alternative splicing patterns of TAF1 mRNAs in XDP and control brains. The impact of the disease-associated SINE-VNTR-Alu on alternative splicing of microexon 34' was further investigated in cellular assays. Subsequently, microexon 34' incorporation was explored by RT-PCR and Nanopore long-read sequencing of TAF1 mRNAs from XDP and control brains tissues. Using cell-based splicing assays, we demonstrate that presence of the disease-associated SINE-VNTR-Alu does not affect the inclusion of microexon 34'. In addition, we show that (1) microexon 34'-containing TAF1 mRNAs are detected at similar levels in XDP as in controls and that (2) the architecture of TAF1 transcripts is remarkably similar between XDP and controls brains. These results indicate that microexon 34' incorporation into TAF1 mRNA is not affected in XDP brains. Our findings shift the current paradigm of XDP by discounting alternative splicing of TAF1 microexon 34' as the molecular basis for this disease.

Promise and challenges of dystonia brain banking: establishing a human tissue repository for studies of X-Linked Dystonia-Parkinsonism.

X-Linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease affecting individuals with ancestry to the island of Panay in the Philippines. In recent years there has been considerable progress at elucidating the genetic basis of XDP and candidate disease mechanisms in patient-derived cellular models, but the neural substrates that give rise to XDP in vivo are still poorly understood. Previous studies of limited XDP postmortem brain samples have reported a selective dropout of medium spiny neurons within the striatum, although neuroimaging of XDP patients has detected additional abnormalities in multiple brain regions beyond the basal ganglia. Given the need to fully define the CNS structures that are affected in this disease, we created a brain bank in Panay to serve as a tissue resource for detailed studies of XDP-related neuropathology. Here we describe this platform, from donor recruitment and consent to tissue collection, processing, and storage, that was assembled within a predominantly rural region of the Philippines with limited access to medical and laboratory facilities. Thirty-six brains from XDP individuals have been collected over an initial 4 years period. Tissue quality was assessed based on histologic staining of cortex, RNA integrity scores, detection of neuronal transcripts in situ by fluorescent hybridization chain reaction, and western blotting of neuronal and glial proteins. The results indicate that this pipeline preserves tissue integrity to an extent compatible with a range of morphologic, molecular, and biochemical analyses. Thus the algorithms that we developed for working in rural communities may serve as a guide for establishing similar brain banks for other rare diseases in indigenous populations.

SVA insertion in X-linked Dystonia Parkinsonism alters histone H3 acetylation associated with TAF1 gene.

X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease linked to an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This SVA insertion induces aberrant TAF1 splicing and partial intron retention, thereby decreasing levels of the full-length transcript. Here we sought to determine if these altered transcriptional dynamics caused by the SVA are also accompanied by local changes in histone acetylation, given that these modifications influence gene expression. Because TAF1 protein may itself exhibit histone acetyltransferase activity, we also examined whether decreased TAF1 expression in XDP cell lines and post-mortem brain affects global levels of acetylated histone H3 (AcH3). The results demonstrate that total AcH3 are not altered in XDP post-mortem prefrontal cortex or cell lines. We also did not detect local differences in AcH3 associated with TAF1 exons or intronic sites flanking the SVA insertion. There was, however, a decrease in AcH3 association with the exon immediately proximal to the intronic SVA, and this decrease was normalized by CRISPR/Cas-excision of the SVA. Collectively, these data suggest that the SVA insertion alters histone status in this region, which may contribute to the dysregulation of TAF1 expression.

Neuroinflammation and histone H3 citrullination are increased in X-linked Dystonia Parkinsonism post-mortem prefrontal cortex.

Neuroinflammation plays a pathogenic role in neurodegenerative diseases and recent findings suggest that it may also be involved in X-linked Dystonia-Parkinsonism (XDP) pathogenesis. Previously, fibroblasts and neuronal stem cells derived from XDP patients demonstrated hypersensitivity to TNF-α, dysregulation in NFκB signaling, and an increase in several pro-inflammatory markers. However, the role of inflammatory processes in XDP patient brain remains unknown. Here we demonstrate that there is a significant increase in astrogliosis and microgliosis in human post-mortem XDP prefrontal cortex (PFC) compared to control. Furthermore, there is a significant increase in histone H3 citrullination (H3R2R8R17cit3) with a concomitant increase in peptidylarginine deaminase 2 (PAD2) and 4 (PAD4), the enzymes catalyzing citrullination, in XDP post-mortem PFC. While there is a significant increase in myeloperoxidase (MPO) levels in XDP PFC, neutrophil elastase (NE) levels are not altered, suggesting that MPO may be released by activated microglia or reactive astrocytes in the brain. Similarly, there was an increase in H3R2R8R17cit3, PAD2 and PAD4 levels in XDP-derived fibroblasts. Importantly, treatment of fibroblasts with Cl-amidine, a pan inhibitor of PAD enzymes, reduced histone H3 citrullination and pro-inflammatory chemokine expression, without affecting cell survival. Taken together, our results demonstrate that inflammation is increased in XDP post-mortem brain and fibroblasts and unveil a new epigenetic potential therapeutic target.

Defects in the striatal neuropeptide Y system in X-linked dystonia-parkinsonism.

Neuropeptide Y is a novel bioactive substance that plays a role in the modulation of neurogenesis and neurotransmitter release, and thereby exerts a protective influence against neurodegeneration. Using a sensitive immunohistochemical method with a tyramide signal amplification protocol, we performed a post-mortem analysis to determine the striatal localization profile of neuropeptide Y in neurologically normal individuals and in patients with X-linked dystonia-parkinsonism, a major representative of the neurodegenerative diseases that primarily involve the striatum. All of the patients examined were genetically verified as having X-linked dystonia-parkinsonism. In normal individuals, we found a scattered distribution of neuropeptide Y-positive neurons and numerous nerve fibres labelled for neuropeptide Y in the striatum. Of particular interest was a differential localization of neuropeptide Y immunoreactivity in the striatal compartments, with a heightened density of neuropeptide Y labelling in the matrix compartment relative to the striosomes. In patients with X-linked dystonia-parkinsonism, we found a significant decrease in the number of neuropeptide Y-positive cells accompanied by a marked loss of their nerve fibres in the caudate nucleus and putamen. The patients with X-linked dystonia-parkinsonism also showed a lack of neuropeptide Y labelling in the subventricular zone, where a marked loss of progenitor cells that express proliferating cell nuclear antigen was found. Our results indicate a neostriatal defect of the neuropeptide Y system in patients with X-linked dystonia-parkinsonism, suggesting its possible implication in the mechanism by which a progressive loss of striatal neurons occurs in X-linked dystonia-parkinsonism.

Understanding XDP through imaging,pathology,and genetics

The X-linked dystonia-parkinsonism (XDP) is a severe progressive, adult-onset X-linked endemic disorder in Filipinos, which is characterized by dystonic movements that start in the third of fourth decade, and replaced by parkinsonism beyond the 10th year of illness. Understanding the pathophysiology of XDP and development of rational therapies will depend on observations from imaging pathological and genetic studies. In this paper we summarize the results of these studies on patients with XDP. The cranial magnetic resonance imaging shows hy-perintense putaminal rim in both dystonic and parkinsonian stages, and atrophy of the caudate head or putamen in the parkinsonian stage. Neuropathological findings show atrophy of the caudate nucleus and putamen, with mild to severe neuronal loss and gliosis. In the neostriatum, the dystonic phase of XDP shows the involvement of striosomes and matrix sparing, while the later, i.e., p[arkinsonian phase, shows matrix involvement as well. In the dystonic phase, the loss of striosomal inhibitory projections lead to disinhibition of nigral dopaminergic neurons, perhaps resulting in a hyperkinetic state; while in the parkinsonian phase, severe and critical reduction of matrix-based projection may result in extranigral parkinsonism. Genetic sequencing of the XDP critical region in Xq13.1 has revealed an SVA retronsposon insertion in an intron of TAF1. This may reduce neuron-specific expression of the TAF1 isoform in the caudade nucleus, and subsequently interfere with the transcription of many neuronal genes, including DRD2. Findings from imaging, pahtology, and genetics studies are gradually shedding light on the pathophysiology of XDP, which hopefully will lead to mare rational and directed therapies.

Understanding XDP through imaging, pathology, and genetics.

The X-linked dystonia-parkinsonism (XDP) is a severe, progressive, adult-onset, X-linked endemic disorder in Filipinos, which is characterized by dystonic movements that start in the third or fourth decade, and replaced by parkinsonism beyond the 10th year of illness. Understanding the pathophysiology of XDP and development of rational therapies will depend on observations from imaging, pathological, and genetic studies. In this paper we summarize the results of these studies on patients with XDP. The cranial magnetic resonance imaging shows hyperintense putaminal rim in both dystonic and parkinsonian stages, and atrophy of the caudate head or putamen in the parkinsonian stage. Neuropathological findings show atrophy of the caudate nucleus and putamen, with mild to severe neuronal loss and gliosis. In the neostriatum, the dystonic phase of XDP shows the involvement of striosomes and matrix sparing, while the later, i.e., parkinsonian phase, shows matrix involvement as well. In the dystonic phase, the loss of striosomal inhibitory projections lead to disinhibition of nigral dopaminergic neurons, perhaps resulting in a hyperkinetic state; while in the parkinsonian phase, severe and critical reduction of matrix-based projection may result in extranigral parkinsonism. Genetic sequencing of the XDP critical region in Xq13.1 has revealed an SVA retrotransposon insertion in an intron of TAF1. This may reduce neuron-specific expression of the TAF1 isoform in the caudate nucleus, and subsequently interfere with the transcription of many neuronal genes, including DRD2. Findings from imaging, pathology, and genetics studies are gradually shedding light on the pathophysiology of XDP, which hopefully will lead to more rational and directed therapies.

Functional anatomy of the basal ganglia in X-linked recessive dystonia-parkinsonism.

Dystonia is a neurological syndrome characterized by sustained muscle contractions that produce repetitive twisting movements or abnormal postures. X-linked recessive dystonia parkinsonism (XDP; DYT3; Lubag) is an adult-onset disorder that manifests severe and progressive dystonia with a high frequency of generalization. In search for the anatomical basis for dystonia, we performed postmortem analyses of the functional anatomy of the basal ganglia based on the striatal compartments (ie, the striosomes and the matrix compartment) in XDP. Here, we provide anatomopathological evidence that, in the XDP neostriatum, the matrix compartment is relatively spared in a unique fashion, whereas the striosomes are severely depleted. We also document that there is a differential loss of striatal neuron subclasses in XDP. In view of the three-pathway basal ganglia model, we postulate that the disproportionate involvement of neostriatal compartments and their efferent projections may underlie the manifestation of dystonia in patients with XDP. This study is the first to our knowledge to show specific basal ganglia pathology that could explain the genesis of dystonia in human heredodegenerative movement disorders, suggesting that dystonia may result from an imbalance in the activity between the striosomal and matrix-based pathways.