Regional and Compartmental Changes Drive Progressive Patterns of Striatal Degeneration in X‐Linked Dystonia Parkinsonism

Abstract

Objective

Initially described in 1976, X‐linked dystonia parkinsonism (XDP) is a neurodegenerative disease that can be characterized by the presentation of dystonia and parkinsonism symptoms. Although this disease bears some resemblance to other neurodegenerative diseases in terms of symptomatology, the pathological signature of XDP is still unclear, in part because of the limited research that has been conducted. This study aims to delineate in detail the neurochemical changes occurring in the post mortem human XDP striatum and to explore their relevance to clinical symptomatology.

Methods

The profiles of immunohistochemical expression of multiple spiny projection neurons and projection fiber markers was assessed in the post mortem tissue from the basal ganglia of 18 XDP and 10 neurologically normal cases.

Results

We present evidence that degeneration of the XDP striatum follows a dorsoventral gradient, with greater vulnerability in the matrix compartment of the striatum. Considering the available clinical data of the cases investigated, we propose that this pattern of neurodegeneration is more closely associated with the duration of the disease than SINE‐VNTR‐Alu repeat length; however, repeat length is inversely correlated with XDP symptom onset and, therefore, influences the clinical timeline.

Interpretation

Our findings provide a novel perspective on the pattern and degree of degeneration of the XDP striatum. We propose that the matrix compartment is more vulnerable to disease impact relative to striosomes than initially thought, and the pattern of striatal degeneration may be indicative of the breakdown of a wider cortico‐basal ganglia neural circuit over the duration of the disease. ANN NEUROL 2026;100:751–767

X‐linked dystonia parkinsonism (XDP) is a hereditary neurodegenerative disease endemic to the island of Panay, Philippines. ¹ Since its initial description in 1976, numerous studies have documented various clinical and pathological features of XDP. ¹ , ² , ³ , ⁴ , ⁵ , ⁶ , ⁷ , ⁸ The symptoms of XDP can manifest themselves as a combination of both dystonia and parkinsonian features, with the latter symptom group overlapping closely with the cardinal symptoms of Parkinson's disease (PD). Notably, and echoing the etiology of Huntington's disease (HD), the variable symptom profile of XDP stems from a monogenic origin. ⁹ , ¹⁰ Of particular interest is the variable hexameric repeat length in the SINE‐VNTR‐Alu (SVA)‐type retrotransposon of the TATA‐box binding protein associated factor 1 (TAF1) gene. ⁹ , ¹⁰ The neuropathological signature of XDP currently centers around the striatum, which becomes severely atrophied over the course of the disease, echoing the pathology of HD. ⁴ , ⁵ , ⁸ However, despite efforts to understand the foundation of XDP, there remain sizeable gaps in our current understanding of disease pathogenesis and the underlying basis of the unique clinical manifestation. Our study aims to address this gap by providing further insight into the neurochemistry of the XDP striatum in the context of the clinical information we have available.

Of particular interest in XDP is the dorsal striatum, which undergoes marked degeneration. ⁴ , ⁵ The dorsal striatum comprises 2 distinct structures: the caudate nucleus (CN) and the putamen. In addition to the structural subdivisions, the striatum can be histochemically divided into 2 molecularly defined compartments. ¹¹ The striosomal compartment is typically identified as having small islands of predominantly high or low expressing neurochemical markers as seen in cross‐sections, and these are parts of the continuous labyrinthine striosomal system that are best seen in 3 dimensions. The striosomes are surrounded by the much larger matrix compartment. These 2 sub‐compartments are functionally distinct and are differentially affected in neurodegenerative disorders. ¹² Postmortem human studies highlight the preferential loss of striosomes in early‐stage HD cases or in HD cases with a history of predominantly mood symptoms. ¹³ , ¹⁴ , ¹⁵ In PD, the dopaminergic system across these sub‐compartments in the non‐human primate model has been reported to be differentially impacted by disease. ¹² , ¹⁶ , ¹⁷ For XDP, the striatum is widely accepted to be impacted over the course of the disease. ² , ⁷ , ⁸ , ¹⁸ With regards to compartmental vulnerability, information is still sparse, but in 1 study, the selective vulnerability of the striosome compartment has been proposed to underpin the presentation of dystonic symptoms. ⁵ It has been hypothesized that as the symptom profile shifts from dystonia to parkinsonism during the course of XDP progression, the matrix compartment is then also lost. ⁵ It was, therefore, suggested that, similar to HD, the differential degeneration in the striosome and matrix compartments of the striatum underpins the manifestation of XDP symptoms.

Here, by examining the neurochemistry of the XDP striatum in the largest cohort of XDP cases to date, this study aimed to expand our understanding of striatal neuronal vulnerability and its impact on disease manifestation and has resulted in a proposed new view of XDP neuropathology.

Methods

Human Tissue Collection and Processing

Postmortem human tissue from a cohort of 18 male XDP and 2 male neurologically normal cases (Table 1) was obtained from the brain bank in the Philippines in collaboration with the Sunshine Care Foundation (Panay, Philippines), the Collaborative Centre for XDP (Boston, MA), and Makati Medical Center (Makati City, Philippines). Eight additional male neurologically normal cases (Table 1) were added to this study from the Neurological Foundation Human Brain Bank at the Centre for Brain Research, University of Auckland, New Zealand. All protocols conducted in this study were approved by the University of Auckland Human Participation Ethics Committee (14/NTA/208) and by the institutional review board at Makati Medical Centre, and all families provided informed consent. XDP diagnosis for each case was confirmed through genetic testing, as previously detailed. ¹⁹ , ²⁰ The neurologically normal and XDP cohorts were matched based on sex, age at death, and postmortem delay (Fig S1).

TABLE 1.

Characteristics of the XDP and Neurologically Normal Cases in This Study

Case number	Diagnosis	Age at death	PMD	Disease duration	Symptom profile	SVA repeat size	Cause of death
SCF18‐001 a	XDP	47	N/A	8	Dystonia	40	Pneumonia
SCF18‐002 a	XDP	54	31	4	Mixed	41	Myocardial infarction
SCF19‐008 a	XDP	65	20	6	Parkinsonism	41	Aspiration pneumonia
SCF19‐010 a	XDP	55	21	6	Mixed	39	Cardiac arrhythmia
SCF19‐012 a	XDP	36	12	10	Mixed	52	Aspiration pneumonia
SCF19‐013 a	XDP	40	23	2	Mixed	41	Dystonia
SCF19‐014 a	XDP	32	12	2	Dystonia	50	Aspiration pneumonia
SCF19‐015 a	XDP	60	15	11	Mixed	36	Sepsis
SCF19‐019 a	XDP	58	7	7	Mixed	38	Septicemia
SCF19‐031 a	XDP	64	24	26	Mixed	48	Pneumonia
SCF19‐032 a	XDP	66	17	9	Mixed	34	XDP
SCF21‐034 a	XDP	55	N/A	14	N/A	40	Asphyxia
SCF21‐036 a	XDP	59	8	25	N/A	40	Cerebral contusion
SCF21‐038 a	XDP	43	15	13	N/A	47	Laryngeal spasm
SCF22‐042 a	XDP	50	16	3	N/A	42	Asphyxia
SCF23‐071 a	XDP	43	N/A	4	N/A	55	Aspiration pneumonia
SCF23‐078 a	XDP	56	33	12	N/A	36	Pneumonia
SCF24‐102 a	XDP	60	19	17	N/A		Aspiration pneumonia
Mean ± SD		52.39 ± 10.14	18.20 ± 7.49
H157 b	Neurologically normal	66	15	N/A	N/A	N/A	Ischemic heart disease
H160 b	Neurologically normal	77	23	N/A	N/A	N/A	Ischemic heart disease
H184 b	Neurologically normal	35	20	N/A	N/A	N/A	Electrocution
H191 b	Neurologically normal	77	25	N/A	N/A	N/A	Ischemic heart disease
H193 b	Neurologically normal	71	23	N/A	N/A	N/A	Coronary atherosclerosis
H240 b	Neurologically normal	73	26.5	N/A	N/A	N/A	Ruptured aneurysm
H242 b	Neurologically normal	51	19.5	N/A	N/A	N/A	Coronary atherosclerosis
H245 b	Neurologically normal	63	20	N/A	N/A	N/A	Asphyxia
SCF21‐035C a	Neurologically normal	40	19.3	N/A	N/A	N/A	Cardiorespiratory arrest
SCF22‐054CM a	Neurologically normal	51	23	N/A	N/A	N/A	Acute myocardial infarction
Mean ± SD		60.40 ± 15.27	21.40 ± 3.33

Details include case numbers, diagnosis, age at death (years), postmortem delay (hours), disease duration (years), dominant symptom profile before death, SVA repeat size, and cause of death. All cases used in this study were male. Cases were characterized as presenting with dystonia, parkinsonism, or a mix of both dystonia and parkinsonism. Unavailable case information is designated as N/A.

PMD = postmortem delay; SD = standard deviation; SVA = SINE‐VNTR‐Alu; XDP = X‐linked dystonia parkinsonism.

^a Tissue sourced from the brain bank in the Philippines.

^b Tissue sourced from the Neurological Foundation Human Brain Bank (NZ).

Free‐Floating Peroxidase Immunohistochemistry

Right hemisphere perfusion‐fixation and dissection of human brain tissue used in this study were conducted according to the protocols fully described in Waldvogel et al ²¹ and Fernandez‐Cerado et al. ¹⁹ 3′3 Diaminobenzidine (DAB) immunohistochemistry and cresyl violet staining protocols were conducted on 70μm‐thick sections of striatum. ²² , ²³ Antibody details are summarized in Table S1.

Image Acquisition and Analysis

Images of the immunolabeled sections were acquired using an Olympus SlideView VS200 slide scanner without illumination or flat‐field correction. Automatic white balance was applied at acquisition, and no subsequent image processing or post‐acquisition normalization was performed. Optical density, as a surrogate measure of protein expression, for the proteins of interest across the dorsal striatum was measured in QuPath (v0.4.4). ²⁴ The wand tool was used to manually annotate the boundaries of the CN, putamen, in addition to the striosome and matrix sub‐compartments of the dorsal striatum. The ventral border of the dorsal striatum was defined as the inferior limit of the internal capsule. ²⁵ Images were inspected for folds or incomplete regions of interest and were excluded, where necessary, from the respective QuPath analysis (denoted by blacked‐out cells in Table S2). Cases with no immunohistochemically identifiable striosomes were excluded from the respective statistical tests (denoted by an asterisk in a grayed‐out cell in Table S2). The mean intensity measurement for each annotation was averaged to provide a mean value for each case.

Statistical analysis was conducted in GraphPad Prism (v9.0) and presented as mean ± standard deviation. As neither cohort was normally distributed, and non‐parametric testing was conducted. Comparisons between neurologically normal and XDP groups were estimated using a 2‐tailed Mann–Whitney test. All p‐values remained statistically significant after applying the 2‐stage step up method of Benjamini, Krieger, and Yekutieli method, with a false discovery rate of 0.05 (Table S3). Correlations between optical density values and either disease duration, SVA repeat length, age at death, or postmortem delay were conducted using Spearman's test and interpreted according to the r‐ and p‐values (Table S4).

Results

The dorsal striatal matrix compartment was characterized in the neurologically normal cases to express higher calbindin⁺ immunostaining intensity  coupled with lower immunostaining for dopamine‐ and cAMP‐regulated phosphoprotein 32 kDa (DARPP‐32)⁺ and enkephalin⁺. Correspondingly, striosomes were differentiated from the matrix in the normal cohort as regions of lower calbindin⁺ staining, greater DARPP‐32⁺, and greater enkephalin⁺ immunoreactivity.

Calbindin Expression Is Reduced in the XDP CN and Putamen

Calbindin, a spiny projection neuron (SPN) marker, ²⁶ is typically enhanced in the matrix compartment of a neurologically normal human striatum (see Fig 1A, B). Consequently, striosomes are typically identified as regions of faint immunoreactivity surrounded by heightened matrix staining. In the XDP striatum, alterations in the calbindin⁺ expression profile were identifiable in a case with a disease duration of 2 years (see Fig 1C), with a staining gradient consisting of lower immunoreactivity in the most superior aspect of the dorsal striatum compared to enhanced staining in the ventral striatum. This dorsal‐to‐ventral gradient was more pronounced in cases with disease durations of more than 8 years, where the ventral striatum exhibits greater calbindin staining relative to the dorsal striatum (see Fig 1E–H). In the XDP case with a 26‐year disease duration, the dorsal striatum presented with a flattened medial outline and an almost complete depletion of calbindin⁺ immunoreactivity, resulting in a pronounced dorsoventral depletion gradient (see Fig 1H–h″).

FIGURE 1.

Matrix‐specific vulnerability of calbindin protein expression in the XDP striatum. (A–H) Representative photomicrographs illustrating striatal calbindin immunoreactivity in (A, B) neurologically normal tissue and (C–H) 6 XDP cases with varying disease durations. The respective case numbers are listed in the top right corner and the dashed line denotes the border between the dorsal and ventral striatum. (a′–h″) Higher power images of striosomes, which are denoted by white asterisks and black dashed line borders, with the surrounding matrix of the dorsal CN. (A–b″) Calbindin exhibits enhanced immunoreactivity in the matrix compartment, with fainter striosomal staining. (C,D) SCF19‐014, an XDP case with a short 2‐year disease duration, and SCF 23–071 (4‐year disease duration) present with relatively variable degrees of calbindin⁺ staining throughout the striatum. (c′, d′) Striosomes in the superior aspect of the dorsal CN are more pronounced with greater immunoreactivity relative to the surrounding matrix. (c″, d″) Striosomes of the inferior aspect of the dorsal CN appear as pale zones of immunoreactivity surrounded by an enriched matrix. (E, F) SCF18‐001 (8‐year disease duration) and SCF19‐015 (11‐year disease duration), exhibits a dorsal‐to‐ventral calbindin⁺ intensity gradient with the ventral region of greatest intensity. (e′ –f″) Calbindin⁺ immunoreactivity appears significantly reduced in the matrix and is enhanced in the striosomes. (G, H) The intensity gradient remains evident in the striatum of XDP cases (G) SCF21‐036 (25‐year disease duration) and (H) SCF19‐031 (26‐year disease duration). (G–g″) Calbindin⁺ striosomes are minimally present throughout the striatum of the 25‐year disease duration XDP case. (H–h″) The CN of the 26‐year disease duration case presents with a flat medial border and no identifiable calbindin⁺ striosomes. (I–N) Densitometric analysis of calbindin⁺ immunoreactivity was conducted in the dorsal striatum of the XDP and neurologically normal cohort. (I) Calbindin⁺ immunoreactivity was identified to significantly reduce by 51.41% (p = 0.011) in the XDP CN (n = 18) compared to neurologically normal cases (n = 9). (J) Matrix‐specific immunoreactivity of the XDP CN (n = 18) was significantly reduced by 74.47% (p = 0.0024) when compared to the neurologically normal cohort (n = 9). (K) No overt changes in calbindin⁺ striosome immunoreactivity (p = 0.24) were found between the XDP (n = 17) and neurologically normal (n = 9) cohort. (L) A 43.27% reduction in calbindin⁺ immunoreactivity across the dorsal putamen (p = 0.027) between the XDP (n = 18) and neurologically normal cohort (n = 9). (M) Calbindin⁺ immunoreactivity in the dorsal XDP putamen matrix (n = 18) was significantly reduced by 72.63% (p = 0.00080) when compared to the neurologically normal cases (n = 9). (N) No significant differences in calbindin⁺ immunoreactivity in the striosomes of the dorsal putamen (p = 0.53) between the XDP (n = 17) and neurologically normal (n = 9) cohort. *p ≤ 0.05, **p ≤ 0.01. CN = caudate nucleus; IC = internal capsule; Put = putamen; XDP = X‐linked dystonia parkinsonism.

Investigations of calbindin⁺ optical density demonstrated an overall 51.41% reduction in the XDP dorsal CN (see Fig 1I, p = 0.011) and a 43.27% reduction in the XDP dorsal putamen (see Fig 1L, p = 0.027) compared to normal cases. Analysis of the XDP matrix compartment demonstrated a 74.47% (see Fig 1J, p = 0.0024) and 72.63% (see Fig 1M, p = 0.00080) reduction in calbindin⁺ optical density, respectively, in the dorsal CN and putamen compared to normal cases. By contrast, striosomal expression levels showed no significant overt differences in calbindin⁺ expression in the striosomes of the XDP cohorts. Therefore, quantitative analysis would suggest that calbindin⁺ expression is diminished in a matrix‐predominant manner in both the dorsal CN and putamen in XDP.

The Reversal of Calbindin Expression within the Striosomes and Matrix Compartments of the XDP Striatum

The pattern of calbindin expression, typically greater in the matrix than in the striosomal regions, is clearly altered in the XDP cohort. In the XDP case with a 2‐year disease duration, the inferior portion of the dorsal striatum retains the typical predominant‐matrix appearance, whereas the matrix compartment of the superior aspect of the dorsal CN presents with diminished calbindin immunoreactivity (see Fig 1c′‐c′′). As such, the striosomes appeared to be more enriched in calbindin expression than the surrounding matrix. In the XDP cases with disease durations ranging from 4 to 25 years, the striosomes present with relatively greater staining intensity, resulting in an apparent reversal of calbindin expression (see Fig 1D–G). Furthermore, calbindin expression was higher at the borders of the striosomes, resulting in the appearance of ring‐like structures in the 4‐, 8‐, and 11‐year disease duration XDP cases. The dorsal striatum of the XDP case with a 26‐year disease duration presented with an overall loss of calbindin⁺ immunoreactivity, resulting in the lack of identifiable sub‐compartments. Comparisons with neighboring DARPP‐32 (see Fig 2H), calcineurin (Fig 3H), enkephalin (see Fig 4H), and substance P (Fig 5H) immunostained sections confirmed the loss of immunohistochemically detectable striosomes. Overall, calbindin⁺ immunostaining would suggest a reversal of staining patterns in the XDP cohort, such that a typical matrix marker in neurologically normal cases is visually enriched in the XDP striosomes. This apparent reversal in staining patterns could stem from the depletion of calbindin in the matrix compartments or calbindin enrichment in the striosomes. Our results demonstrating a significant reduction in the matrix compartment of the dorsal XDP striatum, coupled with no comparable significant changes in the striatal striosomes, would suggest that the matrix sub‐compartment is selectively depleted in the XDP cohort. However, we cannot rule out a scenario where both striosomal enhancement and matrix depletion occur throughout disease progression, with an eventual loss of both striosomes and matrix in the dorsal striatum with a longer duration of disease.

FIGURE 2.

Depletion of DARPP‐32⁺ immunoreactivity across the postmortem human XDP striatum. (A–H) Representative photomicrographs depicting striatal DARPP‐32 immunoreactivity in (A, B) neurologically normal tissue and (C–H) 6 XDP cases with variable disease durations, with respective case numbers in the top right corner. The dashed line marks the border between the dorsal and ventral striatum. (a′–h″) Higher power images of striosomes, which are denoted by white asterisks and black dashed line borders, with the surrounding matrix of the dorsal CN. (A–b″) Normally, DARPP‐32 presents with encapsulated zones of intense staining in the striosomes (denoted by a white asterisk) with moderate immunoreactivity in the surrounding matrix. (a′–b″) DARPP‐32⁺ fiber tracts are denoted by white arrowheads. (C–c″) SCF19‐014, an XDP case with a short 2‐year disease duration, presents with distinct striosomes; however, fewer DARPP‐32⁺ fiber tracts (white arrowhead) are evident. (D–F) A pronounced dorsal‐to‐ventral intensity gradient is evident in XDP cases (D) SCF23‐071 (4‐year disease duration), (E) SCF19‐015 (11‐year disease duration), and (F) SCF19‐034 (14‐year disease duration), with the ventral striatum being the most intense for DARPP‐32⁺. (d′ –f″) Although striosomes remain identifiable, there is a notable reduction in both striosome and matrix immunoreactivity, with little to no identifiable fiber tracts. (G–g″) The dorsal‐to‐ventral intensity gradient remains evident in the striatum of SCF21‐036 (25‐year disease duration). DARPP‐32⁺ striosomes are more readily identifiable toward the inferior aspect of the dorsal CN. (H) SCF19‐031 (26‐year disease duration) presents with a flat medial border of the CN and the intensity gradient is enhanced. (h′–h″) The dorsal CN is devoid of identifiable DARPP‐32⁺ striosomes and appears to be depleted of DARPP‐32 in the matrix. (I–N) Densitometric analysis of DARPP‐32 was conducted in the dorsal striatum of both XDP and neurologically normal cohorts. (I) Overall DARPP‐32⁺ immunoreactivity was reduced by 71.62% (p = 0.0010) in the XDP CN (n = 18) compared to neurologically normal cases (n = 8). (J) Matrix immunoreactivity of the XDP CN (n = 18) was significantly reduced by 71.38% (p = 0.00060) compared to neurologically normal cohort (n = 8). (K) Striosomal‐specific immunoreactivity in the XDP CN (n = 16) was reduced by 64.66% (p = 0.0045) compared to the neurologically normal cohort (n = 8). (L) The dorsal XDP putamen (n = 18) presents with a 60.87% reduction (p = 0.0021) in immunoreactivity compared to the neurologically normal cohort (n = 8). (M) DARPP‐32⁺ matrix immunoreactivity in the dorsal putamen was reduced by 57.36% (p = 0.00080) in XDP (n = 18) compared to neurologically normal cohort (n = 8). (N) Putamen striosomal immunoreactivity (p = 0.037) was reduced by 57.14% in the XDP (n = 13) compared to the neurologically normal (n = 7) cohorts. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. CN = caudate nucleus; DARPP‐32 = dopamine‐ and cAMP‐regulated phosphoprotein 32 kDa; IC = internal capsule; Put = putamen; XDP = X‐linked dystonia parkinsonism.

FIGURE 3.

Calcineurin⁺ expression is lowered in the matrix compartment of the XDP striatum. (A–H) Representative photomicrographs of striatal calcineurin⁺ immunoreactivity in (A, B) neurologically normal tissue and (C–H) 6 XDP cases with varying disease durations. The dashed line demarcates the boundary between the dorsal and ventral striatum. The respective case numbers are listed in the top right corner. (a′–h″) Higher power images of striosomes, which are denoted by white asterisks and black dashed line borders, with the surrounding matrix of the dorsal CN. (A–B) Calcineurin labelling is homogenously distributed throughout the neurologically normal striatum. (a′–b″) As such, striosomes and matrix compartments are immunohistochemically indistinguishable. (C) Striatal calcineurin expression of SCF19‐014, an XDP case of 2‐year disease duration, presents with an overall similar intensity to that of a neurologically normal striatum despite subtle changes in immunoreactivity patterns in the dorsal striatum. (c′) In the superior aspect of the dorsal CN, calcineurin⁺ striosomes can be identified against the surrounding fainter matrix staining. (c″) In the inferior aspect of the dorsal CN, the striatal sub‐compartments present with a similar degree of calcineurin expression. (D–G) XDP cases (D) SCF23‐071 (4‐year disease duration), (E) SCF18‐001 (8‐year disease duration), SCF19‐015 (11‐year disease duration), and SCF21‐036 (25‐year disease duration) present with an overtly altered calcineurin⁺ immunohistochemical profile, with rich striosomal calcineurin immunoreactivity surrounded by a paler matrix compartment. (H) SCF19‐031 (26‐year disease duration) presents with significant depletion of calcineurin in the dorsal striatum and a gradational increase of staining in the ventral striatum. (h′–h″) No identifiable calcineurin⁺ striosomes are present in the dorsal CN. (I–L) Densitometric analysis of calcineurin⁺ immunoreactivity within the dorsal striatum of the XDP and the neurologically normal cohort was conducted. (I) Compared to the neurologically normal cohort (n = 8), the XDP CN (n = 18) presents with a comparable degree of calcineurin⁺ immunoreactivity (p = 0.94). (J) Investigating the differential staining in the sub‐compartments of the XDP CN, the matrix (n = 18) presents with a 41.28% reduction in calcineurin⁺ optical density (p = 0.0054) when compared to the striosomes (n = 17). (K) No overt reductions were observed in overall calcineurin⁺ immunoreactivity (p = 0.81) in the dorsal XDP putamen (n = 18) compared to the neurologically normal cases (n = 8). (L) Calcineurin⁺ matrix immunoreactivity of the dorsal XDP putamen (n = 18) is significantly reduced by 29.83% (p = 0.037) compared to the XDP striosomes (n = 14). *p ≤ 0.05, ***p ≤ 0.001. AC = anterior commissure; CN = caudate nucleus; IC = internal capsule; Mat. = matrix; Put = putamen; Str. = striosomes; XDP = X‐linked dystonia parkinsonism.

FIGURE 4.

Enkephalin loss in the XDP striatum, with peristriosomal preservation until advanced disease. (A–H) Representative photomicrographs of enkephalin⁺ immunoreactivity are depicted in (A, B) neurologically normal tissue and (C–H) 6 XDP cases of varying disease durations, with the case number listed in the top right corner. (a′–h″) Higher power images of striosomes, which are denoted by white asterisks and black dashed line borders, with the surrounding matrix of the dorsal CN. (A, B) In the neurologically normal striatum, enkephalin is enriched in the ring‐like peristriosomal region, termed the annulus. (a′–b″) The core of the striosome and the surrounding matrix exhibit a relatively lower degree of enkephalin⁺ immunoreactivity compared to the peristriosomal boundary. (C) SCF19‐014, an XDP case with a disease duration of 2 years, presents with more intense immunoreactivity in the ventral striatum. (c′) In the superior aspect of the dorsal CN, greater enkephalin⁺ immunoreactivity is present in the annulus relative to the striosomal core and surrounding matrix. (b″) The faintly stained striosomal core remains identifiable in the inferior aspect of the dorsal CN; however, the immunoreactivity pattern of the surrounding matrix is comparable to that of the annulus. (D–F) Enkephalin⁺ immunoreactivity of (D) SCF23‐071 (4‐year disease duration), (E) SCF19‐015 (11‐year disease duration), and (F) SCF19‐034 (14‐year disease duration) presents in a dorsal‐to‐ventral intensity gradient, with the ventral striatum being the most intense. (d′–f″) The striosomes are clearly delineated with an enriched annulus and a faint striosomal core surrounded by the matrix with considerably lower immunoreactivity. (G–g″) Enkephalin⁺ striosomes remain identifiable toward the inferior aspect of the dorsal CN of SCF21‐036 (25‐year disease duration), but are difficult to delineate in the superior aspect of the dorsal CN. (H–h″) In addition to the flat medial border of the CN, enkephalin appears to be depleted in the dorsal striatum of SCF19‐031 (26‐year disease duration) with no immunohistochemically identifiable striosomes. (I–N) Densitometric analysis of enkephalin⁺ expression in the XDP and neurologically normal dorsal striatum. (I) A statistically significant 39.40% reduction (p = 0.0012) in enkephalin⁺ optical density was observed in the XDP CN (n = 18) compared to the neurologically normal CN (n = 9). (J) Matrix‐specific enkephalin⁺ optical density (p = 0.00040) was reduced by 47.39% in the XDP (n = 18) CN when compared to the neurologically normal (n = 9) CN. (K) Striosomes of the XDP CN (n = 18) presented with a 34.14% reduction in enkephalin⁺ optical density (p = 0.0035) compared to the neurologically normal cases (n = 9). (L) The XDP dorsal putamen (n = 18) presented with a 46.31% reduction in enkephalin⁺ immunoreactivity (p = 0.0062) when compared to the neurologically normal (n = 9) putamen. (M) A 48.59% reduction in matrix‐specific enkephalin⁺ immunoreactivity (p = 0.0075) was observed between the XDP (n = 18) and neurologically normal (n = 9) putamen. (N) Striosomal enkephalin⁺ immunoreactivity in the putamen was not significantly different (p = 0.053) between the XDP (n = 14) and neurologically normal (n = 9) cohorts. *p ≤ 0.05, **p ≤ 0.01. AC = anterior commissure; CN = caudate nucleus; IC = internal capsule; Put = putamen; XDP = X‐linked dystonia parkinsonism.

FIGURE 5.

Expression profile of substance P is altered in the XDP striatum. (A–D) Representative photomicrographs of striatal substance P⁺ immunoreactivity in (A, B) neurologically normal tissue and (C–H) 6 XDP cases of varying disease durations, with the respective case numbers listed in the top right corner. (a′–h″) Higher power images of striosomes, which are denoted by white asterisks and black dashed line borders, with the surrounding matrix of the dorsal CN. (A–a′, B–b′) In the neurologically normal case, substance P is diffusely expressed throughout the striosomes and matrix of the dorsal striatum. (a″, b″) Toward the inferior aspect of the dorsal CN, striosomes appear as faintly immunoreactive zones surrounded by a relatively darker matrix. (C) SCF19‐014 (2‐year disease duration) present with variable substance P⁺ staining between the dorsal and ventral striatum. (c′) Substance P⁺ striosomes in the dorsal CN can be identified against a paler surrounding matrix. (c″) In the inferior aspect of the dorsal CN, striosomes appear as pale zones of immunoreactivity surrounded by a substance P‐enriched matrix. (D) SCF23‐071 (2‐year disease duration) presents with overall faint substance P⁺ immunoreactivity. (d′–d″) However, substance P⁺ striosomes can be identified as faintly darker patches against a relatively fainter matrix compartment. (E–F) XDP cases (E) SCF18‐001 (8‐year disease duration) and (F) SCF19‐015 (11‐year disease duration) presents with consistently identifiable substance P⁺ striosomes surrounded by a faintly stained matrix compartment. (G–h″) Longer disease duration XDP cases (G) SCF21‐036 (25‐year disease duration) and SCF19‐031 (26‐year disease duration) presents with an almost complete depletion of substance P⁺ immunoreactivity throughout the striatum and (g′–h″) no identifiable striosomes. (I) Densitometric analysis reveals no statistically significant differences in substance P⁺ optical density (p = 0.17) in the dorsal XDP CN (n = 18) compared to the neurologically normal CN (n = 10). (J) In the XDP cohort, there was a 24.86% difference in substance P⁺ optical density (p = 0.0031) between the striosomes (n = 16) and matrix (n = 18) compartments of the XDP CN. (K) No significant differences in (G) substance P⁺ optical density (p = 0.17) were identified between the XDP (n = 18) and neurologically normal (n = 10) dorsal putamen. (L) The matrix compartment (n = 18) of the XDP putamen presented with 28.74% lower immunoreactivity levels of substance P (p = 0.0076) when compared against the striosome compartments (n = 8). CN = caudate nucleus; IC = internal capsule; Mat. = matrix; Put = putamen; Str. = striosomes; XDP = X‐linked dystonia parkinsonism.

Reduced Expression of DARPP‐32 in the XDP Dorsal Striatum

In the neurologically normal dorsal striatum, DARPP‐32, another SPN marker, ²⁶ presents with heightened staining levels within the striosomes relative to the matrix and fiber bundles. This pattern of heightened DARPP‐32⁺ staining in the striosomes is more evident in the normal CN than in the putamen. By contrast, whereas this pattern of DARPP‐32⁺ expression is preserved in the XDP cohort, there is a visually apparent reduction in overall staining intensity in XDP. Furthermore, although DARPP‐32⁺ fiber bundles are identifiable in the XDP case with a 2‐year disease duration (see Fig 2c″), these fiber bundles are widely absent in the majority of the XDP cohort relative to the neurologically normal cases. The XDP cohort demonstrates an overall 71.62% (see Fig 2I, p = 0.0010) and 60.87% (see Fig 2L, p = 0.0021) reduction in DARPP‐32⁺ optical density within the XDP CN and putamen, respectively. The degree of protein expression is notably reduced in cases with longer disease duration, as evidenced by the drastic depletion of DARPP‐32 in the dorsal striatum of XDP cases with a 25‐year and 26‐year disease duration. Interestingly, the ventral striatum retains enriched DARPP‐32⁺ immunoreactivity. As a result, the XDP striatum presents with a dorsal‐to‐ventral depletion gradient, where DARPP‐32⁺ expression is greater in the ventral striatum compared to the dorsal striatum.

Significant Reduction of DARPP‐32⁺ Expression in Both the Matrix and Striosomes of the XDP Caudate Nucleus

DARPP‐32 immunolabels both sub‐compartments of the neurologically normal striatum, but striosomes can be identified as zones of enriched DARPP‐32⁺ immunoreactivity. Evidence of DARPP‐32 depletion in the XDP striatum is more prominent within the XDP case with a 4‐year or longer disease duration (see Fig 2D–H) compared to the XDP case with a 2‐year duration (see Fig 2C). It follows that DARPP‐32⁺ immunoreactivity in the matrix compartment, specifically, appears more diminished in XDP cases with a disease duration ranging from 4 to 26 years. The XDP CN and putamen matrix compartments present with a 71.38% (see Fig 2J, p = 0.00060) and 57.36% (see Fig 2M, p = 0.00080) loss in DARPP‐32⁺ optical density, respectively. DARPP‐32 depletion is not confined to the matrix region, but can also be observed in the striosomes. Striosomes within the XDP cases with a 4 to 25‐year disease duration appear diminished, but remain identifiable. Noticeably, striosomes toward the most superior part of the CN appear to be fainter in intensity when compared to the striosomes within more inferior parts of the dorsal CN. In marked contrast, there is a complete loss of DARPP‐32⁺ identifiable striosomes in the XDP case with a 26‐year disease duration. Analysis of DARPP‐32⁺ expression in the striosomes reveals a 64.66% loss within the XDP CN (see Fig 2K, p = 0.0045) and a 57.14% loss in the XDP putamen (see Fig 2N, p = 0.037). Our data suggests a loss of DARPP‐32 expression in both the matrix and striosome striatal compartments in XDP.

Calcineurin Is Homogenously Expressed in the Normal Striatum but Reduced in the XDP Striatal Matrix

Calcineurin, another SPN marker, ²⁷ immunolabels both the matrix and striosomes, resulting in a homogenous staining pattern, which is lost in the XDP cohort (see Fig 3A‐H). On first glance, the XDP case with a 2‐year disease duration presents with a homogenous striatal calcineurin distribution with no overtly identifiable sub‐compartments. However, closer inspection of the superior aspect of the dorsal CN reveals subtle diminishment of matrix immunostaining, resulting in defined calcineurin⁺ striosomes. In contrast, the inferior aspect of the dorsal CN retains a more homogeneous staining pattern with no identifiable sub‐compartments. The distribution of calcineurin is notably altered in the cases with a disease duration of 4 to 25 years, with easily identifiable calcineurin‐rich striosomes standing out against a more faintly immunostained surrounding matrix. The ventral striatum presents with a more homogenous distribution of calcineurin. The XDP case with a disease duration of 26‐years presents with reduced calcineurin⁺ immunoreactivity in the dorsal striatum and no identifiable immunohistochemically defined striosomes. The ventral striatum presents with relatively higher calcineurin immunoreactivity, resulting in an overall dorsoventral gradient. Although an apparent redistribution of calcineurin expression was noted in the XDP striatum, densitometric analysis reveals no significant alterations in overall calcineurin⁺ optical density of either the XDP CN or putamen. However, within the XDP cohort, the expression of calcineurin was lower in the matrix compared to the striosomes, with 41.28% and 29.83% lower immunoreactivity levels within the CN (see Fig 3J, p = 0.0054) and putamen (see Fig 3L, p = 0.037), respectively. Altogether, although homogenously expressed in the neurologically normal striatum, calcineurin in the XDP striatum is remodeled during disease pathogenesis, resulting in a notably lower expression of calcineurin within the matrix region of the XDP striatum.

Enkephalin Loss in the XDP Striatum, with Peristriosomal Pattern Preservation until Advanced Disease

In the neurologically normal striatum, enkephalin, a neuropeptide released by SPNs, ¹¹ presents as enriched ring‐like structures with a pale core, demarcating the peristriosomal boundary, which can be discriminated from the surrounding matrix. As per the previously investigated immunohistochemical markers described above, lower enkephalin⁺ immunoreactivity is present throughout the XDP dorsal striatum in comparison to the ventral striatum, resulting in the presence of a dorsoventral staining gradient. The XDP case with a 2‐year disease duration presents with typical enkephalin distribution in the superior aspect of the dorsal striatum. Specifically, the superior aspect of the dorsal striatum contains enriched peristriosomal boundaries around faint striosome cores against a lower‐staining matrix compartment (see Fig 4c′). By contrast, toward the inferior aspect of the dorsal striatum, immunoreactivity of the peristriosomal boundary and the surrounding matrix appears comparable to each other, resulting in the identification of only the faint striosome core (see Fig 4c″). In the XDP cases with a disease duration ranging from 4 to 14 years, albeit fainter, enkephalin immunoreactivity presents with the typical enriched peristriosomal boundary surrounding a faint core and matrix compartment. Although striosomes remain identifiable in the XDP case with a 25‐year disease duration, there is a loss of the typical enkephalin⁺ enriched peristriosomal boundary staining pattern. In the 26‐year disease duration XDP case, there is a depletion of enkephalin and loss of immunohistochemically identifiable striosomes. Inspection of the XDP cohort suggests that the peristriosomal architecture of enkephalin is relatively well preserved until advanced disease durations. However, overall enkephalin⁺ immunoreactivity is notably diminished in XDP cases with a disease duration of 4 years or more. The dorsal XDP CN demonstrates a 39.40% reduction in enkephalin⁺ optical density (see Fig 4I, p = 0.0012), which could be attributed to a 47.39% and 34.14% reduction in the XDP matrix (see Fig 4J, p = 0.00040) and striosome (see Fig 4K, p = 0.0035) compartments, respectively. The XDP putamen demonstrates a 46.31% reduction in enkephalin⁺ expression (see Fig 4L, p = 0.0062), which is attributed to the 48.59% loss of matrix‐specific enkephalin⁺ immunoreactivity (see Fig 4M, p = 0.0075) as no significant changes in enkephalin expression were noted in the striosomes of the XDP putamen.

Differential Expression of Substance P in the XDP Striatum

Substance P (SP), a neuropeptide released by SPNs, ¹¹ which presents relatively homogenously throughout the neurologically normal striatum. However, there is a subtle difference in staining intensity, such that the matrix in the inferior aspect of the dorsal striatum demonstrates a greater degree of staining relative to the fainter striosomes, which is virtually impossible to delineate and quantify consistently across cases (see Fig 5a′–b″). Despite differences in SP staining intensity, in the 2‐year disease duration XDP case, there are regions of differential SP retention, resulting in easily identifiable sub‐compartments (see Fig 5C). In the superior aspect of the dorsal striatum, the striosomes appear as SP‐enriched regions surrounded by a depleted matrix compartment. In contrast, striosomes at the inferior aspect of the dorsal striatum present as focally depleted pockets surrounded by enhanced matrix immunoreactivity. SP expression in the XDP cases with a disease duration of 4 to 11 years becomes more consistent as striosomes present with enriched SP⁺ immunoreactivity throughout the entire dorsal striatum. However, it is evident that striosomes in the superior aspect of the dorsal CN appear to be fainter (see Fig 5e′, f′) than striosomes toward the inferior portion of the dorsal CN (see Fig 5e″, f″). In line with the previously investigated immunohistochemical markers, the 25‐year and 26‐year disease duration XDP case presents with an almost complete depletion of SP⁺ immunoreactivity in the dorsal striatum. Although there are no statistically significant losses or changes in SP⁺ immunoreactivity in the CN and putamen of the XDP cohort, it is visually evident that the pattern of SP distribution is variable across the XDP cohort. When compared to the striosomal compartment, the matrix compartment presents with a 24.86% and 28.74% lower staining intensity in the XDP CN (see Fig 5J, p = 0.0031) and putamen (see Fig 5L, p = 0.0076). Therefore, although SP is homogeneously expressed throughout the neurologically normal striatum, which leaves the striosomes and matrix difficult to discriminate, the XDP striatum demonstrates a redistribution of SP expression, resulting in differential striosome and matrix expression.

Striatal Dysfunction Is More Closely Associated with Disease Duration Than SVA Repeat Length

Our investigation of the XDP striatum demonstrated that the pattern of neurochemical alterations in the dorsal striatum is likely to be associated with disease duration. Of the SPN markers investigated in this study, our correlative analysis demonstrated a moderate negative correlation (Fig S2A, r = −0.56, p = 0.016) between the optical density of DARPP‐32 in the XDP striatum and duration of disease. Calbindin expression in the XPD striatum was also noted to demonstrate a moderate negative relationship (Fig S2B, r = −0.51, p = 0.029) with disease duration. In contrast, no significant relationship was observed between calcineurin expression and disease duration. The optical density of enkephalin (Fig S2D, r = −0.47, p = 0.049), but not SP, demonstrated a weak negative relationship with disease duration. Correlative analysis revealed no significant associations between the expression profiles of our proteins of interest with either SVA repeat length, age at death, or postmortem delay (Table S5). However, it is important to note that SVA repeat length is a modifying factor of disease duration. As our XDP cohort consists predominantly of cases that presented with a mixture of dystonic and parkinsonism symptoms at death (Table 1), the relationship between the striatal neurochemical profile and corresponding symptom profile remains unknown. Altogether, these findings point toward a stronger association between the striatal expression profiles of DARPP‐32, calbindin, and enkephalin with disease duration compared to any other clinical variables available for comparison in this study.

Discussion

Our study investigating the pathological signature of the postmortem human XDP striatum has demonstrated a clear dorsoventral degenerative gradient of abnormality in the striatum, with the greatest vulnerability of the CN from dorsal to ventral, and a preservation of the ventral striatum as evidenced by immunostaining of multiple striatal markers. Our results, summarized in Figure 6, further point to a considerable vulnerability of the matrix neurochemical compartment of the striatum to XDP, relative to that of the striosomal compartment. We propose that the degree of striatal degeneration progresses with advancing disease duration and, importantly, that such correlations were not evident for the other clinical features that we tracked, including SVA repeat length, age at death, or postmortem delay.

FIGURE 6.

Schematic summarizing the significant cellular changes observed in the XDP striatum. All 3 spiny projection neuron markers (calbindin, DARPP‐32, and calcineurin) and both neuropeptide markers (enkephalin and substance P) were significantly altered in the matrix compartment of the XDP striatum compared to neurologically normal control cases. DARPP‐32 = dopamine‐ and cAMP‐regulated phosphoprotein 32 kDa; XDP = X‐linked dystonia parkinsonism.

Striatal degeneration is a feature of many neurodegenerative diseases, including HD. One of the characteristic pathological features of the HD striatum is the dorsomedial to ventrolateral degeneration pattern. ²⁸ We found that the XDP striatum also degenerates along a dorsoventral gradient with advancing disease duration. This gradient was evidenced by subtle distribution changes in immunostaining at the most superior aspect of the dorsal CN, which could be identified in an XDP case with a disease duration of only 2 years, where the matrix compartments were diminished relative to the striosomes. This differential immunostaining pattern subsequently encompassed the entire dorsal CN, which can be identified by 11‐years disease duration. In contrast to and irrespective of disease duration, immunodetected expression patterns of many striatal proteins and peptides were consistently preserved to a greater extent in the ventral XDP striatum, similar to the patterns observed in HD.²⁸ The contrasting preservation of protein expression aligns well with the functional roles attributed to the dorsal and ventral striatum. On one hand, the dorsal striatum is thought to be primarily involved in the regulation of motor and cognitive functions and forms a circuit with the globus pallidus, substantia nigra pars compacta, and the overlying cortical mantle ²⁹ , ³⁰ On the other hand, the ventral striatum is interconnected with the ventral pallidum and cortical regions, modulating the limbic system. ³⁰ The topographical organization of the cerebral cortex is preserved across the striatum. ²⁹ Therefore, this dorsoventral gradient of degeneration could reflect wider dysfunction in the corresponding cortico‐basal ganglia neural circuits and could suggest a key functional relevance in symptom manifestation. ²⁹ , ³⁰

In addition to its dorsal and ventral subdivisions, the human striatum can be divided into 2 functionally distinct interdigitating compartments. ¹¹ The differential functions of the striosomes and matrix compartments have been implicated in symptom manifestation of neurodegenerative diseases, namely the established link between striosomal degeneration and mood dysfunction in HD. ¹⁴ In the dorsal striatum of our XDP cohort, the levels of calbindin and DARPP‐32 expression in the XDP matrix were consistently reduced relative to the neurologically normal matrix. Our findings would, therefore, implicate matrix‐predominant vulnerability in the XDP striatum, as opposed to a striosome‐predominant dysfunction that underpins a hyperkinetic dystonia in XDP, as previously reported. ⁵ A different interpretation of these previously reported findings could offer additional insight. We report that calbindin, typically a matrix marker, is significantly lost in the XDP matrix, resulting in the appearance of calbindin‐enriched striosomes against a fainter surrounding matrix (Fig 1C–c″). Consequently, the matrix compartment could be misinterpreted in the XDP striatum. Instead of a striosomal‐loss model, ⁵ interpreted as underpinning the dystonic symptoms, we suggest, as a working hypothesis, that loss of dorsal regions of the matrix compartment, related to sensorimotor and associative functions, could, in part at least, form the anatomical basis for the presentation of motor dysfunction in XDP. Striosomes were significantly depleted with advanced disease duration, as judged by our immunostaining results comparing cases with different disease durations, but this loss was part of an apparent gradient across time in which the matrix abnormalities were detected earlier. Our findings provide visual evidence that striosomes are implicated in disease pathogenesis in a dorsoventral manner, as evidenced by the fainter DARPP‐32⁺, enkephalin⁺, and SP⁺ striosomes in the superior aspect of the dorsal XDP CN. This late decline of the dorsal striosomes might be related to deficits in neurocognitive functions reported for XDP, including attention and executive function. ³¹ In line with the prior conclusion drawn, ⁵ our findings with more cases with varied disease durations do suggest an imbalance in the matrix and striosome pathways. However, reflecting the limbic‐related connections of striosomes and the sensorimotor connections of the matrix compartments, ¹² gradient loss in the matrix compartment could be more relevant to the XDP motor phenotype than initially thought.

Dystonia was initially understood to be an “early stage” XDP symptom, which is replaced by a parkinsonism phase later in disease progression. ³² , ³³ Correspondingly, studies have drawn a link between striatal atrophy and clinical manifestations. ⁵ , ⁷ , ¹⁸ It was proposed that the “earlier” dystonic phase presents with a hyperintense rim of the putamen, ³² no significant striatal atrophy, ³² but neuropathological striosome depletion. ⁵ In the “later” disease stages, the striatum was proposed to be generally atrophied, ⁷ , ¹⁸ with the matrix compartment depleted, which was proposed to underlie the phenotypic replacement of dystonia with parkinsonism symptoms in late‐stage XDP. ⁵ Here, we considered disease duration, symptoms at death, SVA repeat length, age at death, and postmortem delay. Our findings suggested an association between the changes in protein expression and disease duration, especially of DARPP‐32⁺ expression. SVA repeat length shifts symptom onset ²⁰ , ³⁴ and, still under study, ³ a range of genetic and environmental modifiers likely determine the rate of disease progression. Our data may not demonstrate a strong direct association with SVA repeat length, but disease duration is still impacted by SVA repeat length. Therefore, altogether, our findings suggest the dramatic eventual depletion of striosomes might not be the primary driver of parkinsonism symptoms in late XDP, instead, might be a consequence of advancing disease duration.

We note the limitations of this study. In studying a human disease, such as XDP, the most reliable approach continues to be direct research involving living patients, tissue samples, or postmortem human tissue. Postmortem human brain studies provide a cross‐sectional view of end‐stage pathology. As such, firm conclusions regarding temporal changes cannot be made without longitudinal imaging studies or by investigating brain tissue from earlier disease stages, such as from pre‐manifest patients, which are impossible to obtain. Our study, like others, has been limited by the challenge of obtaining postmortem samples that represent a broad clinical spectrum. Future studies are required to resolve the temporal progression of XDP pathophysiology and any potential associations with systemic co‐pathology or vascular alterations. We further note that we used DAB‐based immunohistochemical detection, despite its limitations (Fig S3), because of both the need to use intense staining to detect low‐level antigenicity and the overlap in primary antibody host species, which precluded the ability to fluorescently co‐label these antibodies. In line with prior studies, striosomes and matrix compartments in our cohort were more readily distinguishable in the dorsal CN in comparison to the putamen and ventral striatum. ²⁶ , ³⁵ , ³⁶ Consequently, the identification of striosomes within the dorsal CN was more straightforward compared to the putamen. The difference in striosomal distinction could reflect overall differences in synaptic density throughout the human striatum.³⁷ Therefore, further studies, including those investigating a wider panel of proteins and genes with different approaches for detection will be required to determine the full patterns of abnormality within the striatum and wider basal ganglia‐related circuits. We did not analyze striatal cell bodies, such as SPNs and interneurons, within the striatal regions analyzed, relying on neuropil and fiber labelling to identify compartments and their boundaries. We did not undertake assays of glial populations, vasculature, or white matter changes. These, too, remain potential therapeutic targets.

In conclusion, we have provided detailed evidence for striatal dysfunction in the largest cohort of XDP cases to date. Our neuroanatomical investigation demonstrates a dorsoventral degeneration gradient in the XDP striatum, which is reminiscent of HD. We also present evidence of significant matrix compartment vulnerability within the striatum of XDP cases, with greater loss of neurochemical reactivity with advancing disease duration.

Author Contributions

A.Y.S.T., R.L.M.F., A.M.G., and M.K.S.B. contributed to the conception and design of the study; A.Y.S.T., N.S.S.S.T., C.J.A., S.B.H., E.B.P., G.A.S., C.C.E.D., M.C.A., E.M., C.C., G.P.L., S.V., M.S., C.P.T., M.L.M., M.D., M.A.C. contributed to the acquisition and analysis of data; A.Y.S.T., A.M.G., R.L.M.F., and M.K.S.B., contributed to drafting the text or preparing the figures.

Potential Conflicts of Interest

Nothing to report.

Supporting information

Table S1. List of antibodies and respective conditions used for this study. Details include the primary and secondary antibodies with species, company name, catalogue number, and optimised concentrations.

Table S2. Summary of the identifiable regions in the XDP and neurologically normal cohorts. The tick indicates that striosomes and matrix were immunohistochemically identifiable for that case. An asterisk in a greyed‐out cell indicates that no identifiable striosomes can be identified in that region, and the case was omitted from statistical analysis. Due to tissue artefacts such as folded or incomplete regions of interest, cases (indicated blacked‐out cells) were excluded from analysis.

Table S3. Two‐stage step‐up method of Benjamini, Krieger, and Yekutieli for controlling the false discovery rate within this study. All observed p‐values remained statistically significant (indicated in bold).

Table S4. Statistical cut‐offs used for classification of correlation strengths. Correlations were classified according to Spearman's correlation coefficient r value and deemed statistically significant if p < 0.05.

Table S5. Expression levels of DARPP‐32, calbindin, calcineurin, enkephalin, and substance P are not associated with SVA repeat length, age of death, or post‐mortem delay. Correlative analyses were conducted to investigate associations between the expression of specific markers (DARPP‐32, calbindin, calcineurin, enkephalin, and substance P) with either SVA repeat length, age of death, and post‐mortem delay, with no statistically significant relationships identified. DARPP‐32 = Dopamine and cAMP‐regulated phosphoprotein 32 kDa, XDP = X‐linked dystonia parkinsonism.

Figure S1. Age at death and post‐mortem delay of the neurologically normal and XDP cohorts are not significantly different. Mann–Whitney testing was conducted to assess the potential differences in age at death and post‐mortem delay of the neurologically normal and XDP cohorts. (A) No statistically significant differences in the age at death distribution (p = 0.13) was observed between the neurologically normal (n = 10) or XDP (n = 18) cohort. (B) No statistical difference in the distribution of post‐mortem delay (p = 0.13) was identified between the neurologically normal (n = 10) and XDP (n = 15) cohorts. XDP = X‐linked dystonia parkinsonism.

Figure S2. Striatal DARPP‐32, calbindin, and enkephalin expression is inversely associated with disease duration in XDP cases. Spearman's rank correlation was conducted to investigate the relationship between striatal protein expression and disease duration of the cases studied in the XDP cohort (n = 18). (A) The optical density of DARPP‐32 in the XDP striatum demonstrates a moderately inverse relationship (r = −0.56, p = 0.016) with disease duration. (B) Calbindin expression demonstrates a moderate inverse relationship (r = −0.51, p = 0.029) with disease duration in the XDP striatum. (C) No significant relationships were identified between the optical density of calcineurin⁺ expression and disease duration in the XDP striatum (r = −0.24, p = 0.34). (D) Enkephalin⁺ optical density demonstrates a weak inverse relationship with disease duration (r = −0.47, p = 0.049) in the striatum. (E) No significant relationships were identified between case disease duration and optical density of substance P in the XDP striatum (r = −0.42, p = 0.080). *p ≤ 0.05, DARPP‐32 = Dopamine‐ and cAMP‐regulated phosphoprotein 32 kDa, XDP = X‐linked dystonia parkinsonism.

Figure S3. Correlation between DARPP‐32⁺ integrated intensity values obtained by DAB and fluorescent immunohistochemistry. A Pearson's correlation was conducted to investigate DARPP‐32⁺ integrated intensity values obtained by fluorescent and DAB immunohistochemistry in the pooled neurologically normal and XDP cohort (n = 18). (A) A very strong statistically significant positive correlation (r = 0.80, p < 0.0001) was observed between the fluorescent and DAB DARPP‐32⁺ integrated intensity values in the caudate nucleus. (B) A strong, statistically significant, positive correlation (r = 0.72, p = 0.0010) was observed between fluorescent and DAB DARPP‐32⁺ integrated intensity values in the putamen. DAB = 3,3′‐diaminobenzidine, DARPP‐32 = Dopamine‐ and cAMP‐regulated phosphoprotein 32 kDa, XDP = X‐linked dystonia parkinsonism.

Data Availability

A collection containing the complete supporting materials, including all high‐resolution figures presented in the main text and supplementary materials have been deposited to Figshare (https://doi.org/10.17608/k6.auckland.c.8477985). The collection also includes the exemplar DARPP‐32 analysis project used for quantitative image analysis. Original immunohistochemical image files corresponding to all representative images shown in the manuscript are provided in its unprocessed form, alongside high‐resolution images of cresyl violet‐stained sections used for histological assessment and anatomical verification.