BACKGROUND:Parkinson's disease (PD) and multiple system atrophy (MSA) are two distinct α-synucleinopathies traditionally differentiated through clinical symptoms. Early diagnosis of MSA is problematic, and seed amplification assays (SAAs), such as real-time quaking-induced conversion (RT-QuIC), offer the potential to distinguish these diseases through their underlying α-synuclein (α-Syn) pathology and proteoforms. Currently, SAAs provide a binary result, signifying either the presence or absence of α-Syn seeds. To enhance the diagnostic potential and biological relevance of these assays, there is a pressing need to incorporate quantification and stratification of α-Syn proteoform-specific aggregation kinetics into current SAA pipelines. METHODS:Optimal RT-QuIC assay conditions for α-Syn seeds extracted from PD and MSA patient brains were determined, and assay kinetics were assessed for α-Syn seeds from different pathologically relevant brain regions (medulla, substantia nigra, hippocampus, middle temporal gyrus, and cerebellum). The conformational profiles of disease- and region-specific α-Syn proteoforms were determined by subjecting the amplified reaction products to concentration-dependent proteolytic digestion with proteinase K. RESULTS:Using our protocol, PD and MSA could be accurately delineated using proteoform-specific aggregation kinetics, including α-Syn aggregation rate, maximum relative fluorescence, the gradient of amplification, and core protofilament size. MSA cases yielded significantly higher values than PD cases across all four kinetic parameters in brain tissues, with the MSA-cerebellar phenotype having higher maximum relative fluorescence than the MSA-Parkinsonian phenotype. Statistical significance was maintained when the data were analysed regionally and when all regions were grouped. CONCLUSIONS:Our RT-QuIC protocol and analysis pipeline can distinguish between PD and MSA, and between MSA phenotypes. MSA α-Syn seeds induce faster propagation and exhibit higher aggregation kinetics than PD α-Syn, mirroring the biological differences observed in brain tissue. With further validation of these quantitative parameters, we propose that SAAs could advance from a yes/no diagnostic to a theranostic biomarker that could be utilised in developing therapeutics.

Huntington’s disease (HD) is caused by a CAG repeat expansion in the HTT gene, leading to altered gene expression. However, the mechanisms leading to disrupted RNA processing in HD remain unclear. Here we identify TDP-43 and the N6-methyladenosine (m6A) writer protein METTL3 to be upstream regulators of exon skipping in multiple HD systems. Disrupted nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 occurs in HD mouse and human brains, with TDP-43 also co-localizing with HTT nuclear aggregate-like bodies distinct from mutant HTT inclusions. The binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in the striatum of HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a mechanism underlying alternative splicing in HD. Nguyen et al. identify TDP-43 and METTL3 as key regulators of disrupted RNA splicing in Huntington’s disease, offering insight into how TDP-43 mislocalization and aberrant m6A RNA modification and localization relate to disease pathogenesis.

In 2017 a novel growth factor administration therapy (termed NTCELL®) was trialled for safety and efficacy for Parkinson's disease treatment. NTCELL® administration is the transplantation of encapsulated porcine choroid plexus cells into the putamen. A clinical study demonstrated safety but failed to meet its primary clinical end-point. Here we describe the clinical and neuropathological characteristics of a case who died five years after receiving NTCELL® treatment. Using histochemical and immunohistochemical labelling, we demonstrate that the NTCELL® capsules remain in the striatum but are mainly devoid of cellular contents. A similar pattern of astrogliosis and microgliosis surrounds the capsule placement area to those surrounding the needle track for implanting the capsules. This study is important as it is the first exploration of the long-term clinical and anatomical outcomes of the NTCELL® clinical trial. The results of this study will help inform future studies aimed at regenerating neurons through the implantation of capsules.

Although the genetic basis of Huntington's disease (HD) has been determined, the underlying pathophysiological mechanisms contributing to neurodegeneration remain largely unknown. In recent years, increasing evidence has posited vascular dysfunction as a significant early event in disease pathogenesis; however, these processes remain to be fully elucidated. High-content immunohistochemical screening studies were conducted on HD middle temporal gyrus (MTG) human brain tissue microarrays (TMAs) to investigate various components of the vascular system, including endothelial cells (UEA-1), pericytes (PDGFRβ), vascular smooth muscle cells (αSMA), extracellular matrix components (ECM; collagen IV and fibronectin), and leakage markers (haemoglobin and fibrinogen). Analyses of vascular markers revealed an increase in the number of vessels in the HD TMA cohort which was associated with advancing striatal pathology and earlier symptom onset. Furthermore, our findings highlight the preservation of pericytes, vascular smooth muscle cells, ECM components, and blood-brain barrier integrity in the HD MTG. Collectively, the TMA findings allude to mild vascular remodelling in the temporal cortex which is known to present with a lesser degree of neuronal degeneration in HD.

Despite vulnerability to microstructural tissue damage following mild traumatic brain injury (mTBI), key subcortical brain regions have been overlooked in quantitative susceptibility mapping (QSM) studies. Alterations to tissue composition in the functionally and structurally distinct hippocampal subfields and basal ganglia regions may reflect distinct symptomatology, and better characterisation of these regions is needed to increase our understanding of mTBI pathophysiology. To address this issue, we analysed differences in positive and negative QSM values between 25 males with acute ( < 14 days) sports-related mTBI (sr-mTBI) and 25 age-matched male controls across 10 hippocampal subfields and 16 basal nuclei. Additional variables of interest including age, injury severity, and days since injury at time of the magnetic resonance imaging (MRI) scan were also correlated with both positive and negative susceptibility values. Primary analyses indicated no significant difference in positive susceptibility values between sr-mTBI participants and controls for hippocampal and basal ganglia ROIs. For negative sign values, susceptibility was significantly less negative for sr-mTBI participants in the cornu ammonis 4 (CA4) region only (pFDR = 0.04). In line with the known linear relationship between iron deposition and age in deep grey matter, particularly within the first three decades of life, significant positive relationships were observed between net positive susceptibility and age in the putamen, caudate, red nucleus, parabrachial pigmented nucleus, and ventral pallidum (pFDR < 0.05). Positive relationships were also observed between absolute negative susceptibility values and age in the hippocampal fimbria, caudate, and extended amygdala (pFDR < 0.05), suggesting age-related calcifications in these regions. A negative relationship was observed between absolute negative values and age in the ventral pallidum (pFDR = 0.04), indicating potential changes to myelin content in this region. No significant associations were observed between any other variable and signed susceptibility values. The results of this study contribute to, and extend, prior literature regarding the temporal kinetics of biomagnetic substrates as a function of ageing. Decreased negative susceptibility after mTBI in the CA4 region also suggests potential injury-related effects on myelin content or neuron loss; a particularly interesting finding in light of the well-established vulnerability of cell populations in this region and susceptibility to pathology in chronic traumatic encephalopathy (CTE). The lack of other significant between-group differences suggest that alterations to tissue content may not be quantifiable at the acute stage of injury in subcortical ROIs or may be masked by age-related tissue susceptibility changes as a common feature across all participants in this young cohort. Future research should consider the use of longitudinal study designs to mitigate the influence of these factors. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by a grant from the Health Research Council of New Zealand (HRC), grant \#21/622. The first author (CE) is supported by a Dame Dorothy Winstone Doctoral Completion Award from the Kate Edger Foundation of New Zealand. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Health and Disabilities Ethics Committee (HDEC) gave ethical approval for this work The Auckland University of Technology Ethics Committee (AUTEC) gave institutional approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Ubiquilin 2, encoded by the X-linked UBQLN2 gene, is a ubiquitin-binding quality control protein. Pathogenic UBQLN2 genetic variants cause X-linked dominant amyotrophic lateral sclerosis and/or frontotemporal dementia (ALS/FTD), however, clinical phenotypes from these variants show striking inter- and intra-familial heterogeneity. Further, there are many UBQLN2 variants whose significance to disease is uncertain. Here, we examine the pathogenic potential of UBQLN2 variants reported in individuals with ALS/FTD and their non-symptomatic relatives. Meta-analysis from 27 published studies identified 186 affected individuals and 51 asymptomatic carriers, each harbouring one of 43 unique UBQLN2 coding variants. Features of identified variants, including evolutionary conservation, minor allele frequencies, localisation to protein domains, and in silico predictions of pathogenicity were compiled. Per biological sex, clinical features were compared between UBQLN2 variants segregated by pathogenicity. Pathogenic UBQLN2 variant carriers, most of whom are familial ALS cases, showed a sex-specific difference in age at onset wherein males developed disease on average 18.15 years prior to females (29.54 ± 11.9 versus 47.69 ± 13.4 years, p < 0.0001), with no change in disease duration (p = 0.2091). UBQLN2 variants of uncertain significance showed a bimodal distribution of onset age per sex suggesting a mixture of true benign and true pathogenic variants. In human brain tissue, two male UBQLN2 p.Thr487Ile (ALS-FTD and ALS) cases showed a greater burden of ubiquilin 2 aggregates than a related female case (ALS-FTD). These robust sex-specific differences in ALS/FTD presentation in carriers of pathogenic UBQLN2 variants may improve predictions of ALS/FTD risk in carriers, aiding in diagnosis and disease management.

Evidence has linked head trauma to increased risk factors for neuropathology, including mechanical deformation of the sulcal fundus and, later, perivascular accumulation of hyperphosphorylated tau adjacent to these spaces related to chronic traumatic encephalopathy. However, little is known about microstructural abnormalities and cellular dyshomeostasis in acute mild traumatic brain injury in humans, particularly in the cortex. To address this gap, we designed the first architectonically motivated quantitative susceptibility mapping study to assess regional patterns of net positive (iron-related) and net negative (myelin-, calcium-, and protein-related) magnetic susceptibility across 34 cortical regions of interest following mild traumatic brain injury. Bilateral, between-group analyses sensitive to cortical depth and curvature were conducted between 25 males with acute (<14 d) sports-related mild traumatic brain injury and 25 age-matched male controls. Results suggest a trauma-induced increase in net positive susceptibility focal to superficial, perivascular-adjacent spaces in the parahippocampal sulcus. Decreases in net negative susceptibility values in distinct voxel populations within the same region indicate a potential dual pathology of neural substrates. These mild traumatic brain injury–related patterns were distinct from age-related processes revealed by correlation analyses. Our findings suggest depth- and curvature-specific deposition of biological substrates in cortical tissue convergent with features of misfolded proteins in trauma-related neurodegeneration.

Microglia are the innate immune cells of the brain with the capacity to react to damage or disease. Microglial reactions can be characterised in post-mortem tissues by assessing their pattern of protein expression, or immunophenotypes, and cell morphologies. We recently demonstrated that microglia have a phagocytic immunophenotype in early-stage ALS but transition to a dysfunctional immunophenotype by end stage, and that these states are driven by TAR DNA-binding protein 43 (TDP-43) aggregation in the human brain. However, it remains unclear how microglial morphologies are changed in ALS. Here we examine the relationship between microglial immunophenotypes and morphologies, and TDP-43 pathology in motor cortex tissue from people with ALS and from a TDP-43-driven ALS mouse model. Post-mortem human brain tissue from 10 control and 10 ALS cases was analysed alongside brain tissue from the bigenic NEFH-tTA/tetO-hTDP-43∆NLS (rNLS) mouse model of ALS at distinct disease stages. Sections were immunohistochemically labelled for microglial markers (HLA-DR, CD68, and Iba1) and phosphorylated TDP-43 (pTDP-43). Single-cell microglial HLA-DR, CD68, and Iba1 average intensities, and morphological features (cell body area, process number, total outgrowth, and branch number) were measured using custom image analysis pipelines. In human ALS motor cortex, we identified a significant change in microglial morphologies from ramified to hypertrophic, which was associated with increased Iba1 and CD68 levels. In the rNLS mouse motor cortex, the microglial morphologies changed from ramified to hypertrophic and increased Iba1 levels occurred in parallel with pTDP-43 aggregation, prior to increases in CD68 levels. Overall, the evidence presented in this study demonstrates that microglia change their morphologies prior to immunophenotype changes. These morphological changes may prime microglia near neurons with pTDP-43 aggregation for phagocytosis, in turn triggering immunophenotype changes; first, to a phagocytic state then to a dysfunctional one.

MicroRNAs (miRNAs) are small non-coding RNAs that play crucial roles in post-transcriptional gene regulation. Poly(A) RNA polymerase D5 (PAPD5) catalyzes the addition of adenosine to the 3' end of miRNAs. In this study, we demonstrate that the Yin Yang 1 protein, a transcriptional repressor of PAPD5, is recruited to both RNA foci and protein aggregates, resulting in an upregulation of PAPD5 expression in Huntington's disease (HD). Additionally, we identify a subset of PAPD5-regulated miRNAs with increased adenylation and reduced expression in our disease model. We focus on miR-7-5p and find that its reduction causes the activation of the TAB2-mediated TAK1-MKK4-JNK pro-apoptotic pathway. This pathway is also activated in induced pluripotent stem cell-derived striatal neurons and post-mortem striatal tissues isolated from HD patients. In addition, we discover that a small molecule PAPD5 inhibitor, BCH001, can mitigate cell death and neurodegeneration in our disease models. This study highlights the importance of PAPD5-mediated miRNA dysfunction in HD pathogenesis and suggests a potential therapeutic direction for the disease.

Chronic traumatic encephalopathy (CTE), a neurodegenerative disease associated with repetitive head injuries, is characterised by perivascular hyperphosphorylated tau (p-tau) accumulations within the depths of cortical sulci. Although the majority of CTE literature focuses on p-tau pathology, other pathological features such as glial reactivity, vascular damage, and axonal damage are relatively unexplored. In this study, we aimed to characterise these other pathological features, specifically in CTE p-tau lesion areas, to better understand the microenvironment surrounding the lesion. We utilised multiplex immunohistochemistry to investigate the distribution of 32 different markers of cytoarchitecture and pathology that are relevant to both traumatic brain injury and neurodegeneration. We qualitatively assessed the multiplex images and measured the percentage area of labelling for each marker in the lesion and non-lesion areas of CTE cases. We identified perivascular glial reactivity as a prominent feature of CTE p-tau lesions, largely driven by increases in astrocyte reactivity compared to non-lesion areas. Furthermore, we identified astrocytes labelled for both NAD(P)H quinone dehydrogenase 1 (NQO1) and L-ferritin, indicating that lesion-associated glial reactivity may be a compensatory response to iron-induced oxidative stress. Our findings demonstrate that perivascular inflammation is a consistent feature of the CTE pathognomonic lesion and may contribute to the pathogenesis of brain injury-related neurodegeneration.

Mild traumatic brain injury (mTBI) is a common condition, particularly pervasive in contact sports environments. A range of symptoms can accompany this type of injury and negatively impact people's lives. As mTBI diagnosis and recovery largely rely on subjective reports, more objective injury markers are needed. The current study compared structural brain MRI-T2 relaxometry between a group of 40 male athletes with mTBI within 14 days of injury and 40 age-matched male controls. Voxel-averaged T2 relaxometry within the gray matter was increased for the mTBI group compared to controls (p < 0.001), with statistically significant increased T2 relaxometry particularly in superior cortical regions. Our findings indicate subtle brain abnormalities can be identified in acute mTBI using MRI-T2 relaxometry. These brain abnormalities may reflect inflammation present in the brain and could constitute an objective injury marker to supplement current subjective methods that dominate clinical decisions regarding diagnosis and prognosis. Future research should validate this potential marker with other data types, such as blood biomarkers or histological samples.

Quantitative susceptibility mapping has been applied to map brain iron distribution after mild traumatic brain injury to understand properties of neural tissue which may be related to cellular dyshomeostasis. However, this is a heterogeneous injury associated with microstructural brain changes, and 'traditional' group-wise statistical approaches may lead to a loss of clinically relevant information, as subtle alterations at the individual level can be obscured by averages and confounded by within-group variability. More precise and individualized approaches are needed to characterize mild traumatic brain injury better and elucidate potential cellular mechanisms to improve intervention and rehabilitation. To address this issue, we use quantitative MRI to build individualized profiles of regional positive (iron-related) magnetic susceptibility across 34 bilateral cortical ROIs following mild traumatic brain injury. Healthy population templates were constructed for each cortical area using standardized Z-scores derived from 25 age-matched male controls aged between 16 and 32 years (M = 21.10, SD = 4.35), serving as a reference against which Z-scores of 35 males with acute (<14 days) sports-related mild traumatic brain injury were compared [M = 21.60 years (range: 16-33), SD = 4.98]. Secondary analyses sensitive to cortical depth and curvature were also generated to approximate the location of iron accumulation in the cortical laminae and the effect of gyrification. Primary analyses indicated that approximately one-third (11/35; 31%) of injured participants exhibited elevated positive susceptibility indicative of abnormal iron profiles relative to the healthy population, a finding that was mainly concentrated in regions within the temporal lobe. Injury severity was significantly higher (P = 0.02) for these participants than their iron-normal counterparts, suggesting a link between injury severity, symptom burden, and elevated cortical iron. Secondary exploratory analyses of cortical depth and curvature profiles revealed abnormal iron accumulation in 83% (29/35) of mild traumatic brain injury participants, enabling better localization of injury-related changes in iron content to specific loci within each region and identifying effects that may be more subtle and lost in region-wise averaging. Our findings suggest that individualized approaches can further elucidate the clinical relevance of iron in mild head injury. Differences in injury severity between iron-normal and iron-abnormal mild traumatic brain injury participants identified in our primary analysis highlight not only why precise investigation is required to understand the link between objective changes in the brain and subjective symptomatology, but also identify iron as a candidate biomarker for tissue pathology after mild traumatic brain injury.

Evidence has linked head trauma to increased risk factors for neuropathology, including acute mechanical deformation of the cortical sulcal fundus and, later, perivascular accumulation of hyperphosphorylated tau (p-tau) adjacent to these spaces related to chronic traumatic encephalopathy (CTE). Despite this, little is known about microstructural abnormalities and cellular dyshomeostasis at the acute stage of mild traumatic brain injury (mTBI) in humans, particularly in the cortex. To address this gap in the literature, we designed the first architectonically-motivated quantitative susceptibility mapping (QSM) study to assess regional patterns of positive (iron-related) and negative (myelin-, calcium-, and protein-related) magnetic susceptibility in cortical regions of interest (ROI) following mTBI. Depth- and curvature-specific positive and negative QSM values were compared between 25 males with acute (< 14 days) sports-related mTBI (sr-mTBI) and 25 age-matched male controls across 34 cortical ROIs. Bilateral between-group analyses were conducted on specific ROI curvature bins (crown, bank, and fundus) as well as a combined curvature measure, across 21 cortical depths, for each ROI. Correlations between positive and negative susceptibility were analysed for age, brain injury severity, and the number of days since injury. We observed significant group differences in magnetic susceptibility for depth, curvature, and ROIs. Our results suggest a trauma-induced pattern of iron deposition preferential to superficial, perivascular-adjacent spaces in the sulci of the parahippocampal gyrus. Co-localised decreases in diamagnetism in the same region suggest dual pathology of neural substrates, the biological mechanisms behind which remain speculative. Significant correlations were found between magnetic susceptibility and age, both in ROIs and cortical depths distinct from those showing sr-mTBI-related differences. Little to no relationship was observed between magnetic susceptibility and subjective markers of injury or injury latency. The coherence between our findings and pathognomonic patterns of misfolded proteins in trauma-related neurodegeneration is interesting, which may have implications for the role of brain iron in microstructural cortical tissue damage after a mild brain injury. Further longitudinal research is needed to elucidate the long-term implications of our findings. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by a grant from the Health Research Council of New Zealand (HRC), grant #21/622. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Auckland University of Technology Ethics Committee gave ethical approval for this work The Health and Disabilities Ethics Committee gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

INTRODUCTION:Tau aggregation into neurofibrillary tangles in Alzheimer's disease (AD) is a dynamic process involving changes in tau phosphorylation, isoform composition, and morphology. To facilitate studies of tangle maturity, we developed an image analysis pipeline to study antibody labeling signatures that can distinguish tangle maturity levels in AD brain tissue. METHODS:Using fluorescent immunohistochemistry, we co-labeled AD brain tissue with four antibodies that bind different tau epitopes. Mean fluorescence intensity of each antibody was measured, and spectral clustering was used to identify tangle immunophenotypes. RESULTS:Five distinct tangle populations were identified, and different tangle maturity immunophenotypes were identified with increasing Braak stage. Early tangle immunophenotypes were more prevalent in later affected regions and advanced immunophenotypes were associated with ghost morphology. DISCUSSION:Our findings indicate that tangle populations characterized by advanced tau immunophenotypes are associated with higher Braak stage and more mature morphology, providing a new framework for defining tangle maturity levels using tau antibody signatures. HIGHLIGHTS:Populations of neurofibrillary tangles exist in Alzheimer's disease. The immunophenotype of neurofibrillary tangle populations relates to their maturity. The most advanced immunophenotypes are associated with higher Braak stage. The most advanced immunophenotypes are associated with ghost morphology. The most immature immunophenotypes are associated with later affected regions.

Huntingtin protein, mutated in Huntington’s disease, is implicated in nucleic acid–mediated processes, yet the evidence for direct huntingtin–nucleic acid interaction is limited. Here, we show wild-type and mutant huntingtin copurify with nucleic acids, primarily RNA, and interact directly with G-rich RNAs in in vitro assays. Huntingtin RNA-immunoprecipitation sequencing from patient-derived fibroblasts and neuronal progenitor cells expressing wild-type and mutant huntingtin revealed long noncoding RNA NEAT1 as a significantly enriched transcript. Altered NEAT1 levels were evident in Huntington’s disease cells and postmortem brain tissues, and huntingtin knockdown decreased NEAT1 levels. Huntingtin colocalized with NEAT1 in paraspeckles, and we identified a high-affinity RNA motif preferred by huntingtin. This study highlights NEAT1 as a huntingtin interactor, demonstrating huntingtin’s involvement in RNA-mediated functions and paraspeckle regulation.

Mild traumatic brain injury (mTBI), often called concussion, is a prevalent condition that can have significant implications for people’s health, functioning and well-being. Current clinical practice relies on self-reported symptoms to guide decision-making regarding return to sport, employment, and education. Unfortunately, reliance on subjective evaluations may fail to accurately reflect the resolution of neuropathology, exposing individuals with mTBI to an increased risk of further head trauma. No objective technique currently exists to assess the microstructural alterations to brain tissue which characterise mTBI. MRI-based T2 relaxation is a quantitative imaging technique that is susceptible to detecting fluid properties in the brain and is hypothesised to indicate neuroinflammation. This study aimed to investigate the potential of individual-level T2 relaxometry to evaluate cellular damage from mTBI. 20 male participants with acute sports-related mTBI (within 14 days post-injury) and 44 healthy controls were recruited for this study. Each mTBI participant’s voxel-wise T2 relaxometry map was analysed against healthy control averages using a voxel-wise z-test with false discovery rate correction. Five participants were re-scanned after clinical recovery and results were compared to their acute T2 relaxometry maps to assess reduction in potential neuroinflammation. T2 relaxation times were significantly increased in 19/20 (95 %) mTBI participants compared to healthy controls, in regions including the hippocampus, frontal cortex, parietal cortex, insula, cingulate cortex and cerebellum. Results suggest the presence of increased cerebral fluid in individuals with mTBI. Longitudinal results indicated a reduction in T2 relaxation for all five participants, indicating a possible resolution over time. This research highlights the potential of individual-level T2 relaxometry MRI as a non-invasive method for assessing subtle brain pathology in mTBI. Identifying and monitoring changes in the fluid content in the brain could aid in predicting recovery and developing individualised treatment plans for individuals with mTBI. Future research should validate this measure with other markers of inflammation (e.g. from blood biomarkers) to test whether T2-relaxometry is related to subtle brain inflammation in mTBI. In addition, future research should utilise larger control groups to establish normative ranges and compute robust z-score analyses.

Huntington's disease (HD) is a neurodegenerative disorder that severely affects the basal ganglia and regions of the cerebral cortex. While astrocytosis and microgliosis both contribute to basal ganglia pathology, the contribution of gliosis and potential factors driving glial activity in the human HD cerebral cortex is less understood. Our study aims to identify nuanced indicators of gliosis in HD which is challenging to identify in the severely degenerated basal ganglia, by investigating the middle temporal gyrus (MTG), a cortical region previously documented to demonstrate milder neuronal loss. Immunohistochemistry was conducted on MTG paraffin-embedded tissue microarrays (TMAs) comprising 29 HD and 35 neurologically normal cases to compare the immunoreactivity patterns of key astrocytic proteins (glial fibrillary acidic protein, GFAP; inwardly rectifying potassium channel 4.1, Kir4.1; glutamate transporter-1, GLT-1; aquaporin-4, AQP4), key microglial proteins (ionised calcium-binding adapter molecule-1, IBA-1; human leukocyte antigen (HLA)-DR; transmembrane protein 119, TMEM119; purinergic receptor P2RY12, P2RY12), and indicators of proliferation (Ki-67; proliferative cell nuclear antigen, PCNA). Our findings demonstrate an upregulation of GFAP+ protein expression attributed to the presence of more GFAP+ expressing cells in HD, which correlated with greater cortical mutant huntingtin (mHTT) deposition. In contrast, Kir4.1, GLT-1, and AQP4 immunoreactivity levels were unchanged in HD. We also demonstrate an increased number of IBA-1+ and TMEM119+ microglia with somal enlargement. IBA-1+, TMEM119+, and P2RY12+ reactive microglia immunophenotypes were also identified in HD, evidenced by the presence of rod-shaped, hypertrophic, and dystrophic microglia. In HD cases, IBA-1+ cells contained either Ki-67 or PCNA, whereas GFAP+ astrocytes were devoid of proliferative nuclei. These findings suggest cortical microgliosis may be driven by proliferation in HD, supporting the hypothesis of microglial proliferation as a feature of HD pathophysiology. In contrast, astrocytes in HD demonstrate an altered GFAP expression profile that is associated with the degree of mHTT deposition.

Antigen retrieval is crucial for immunohistochemistry, particularly in formalin-fixed paraffin-embedded brain tissue, where fixation causes extensive crosslinking that masks epitopes. Heat Induced Epitope Retrieval (HIER) reverses these crosslinks, improving access to nuclear and aggregated proteins. We introduce Cyclic Heat-Induced Epitope Retrieval (CHIER), an advanced technique that builds on HIER by incorporating repeated cycles of heating and cooling. CHIER optimises antigen retrieval and significantly improves detection. CHIER is particularly effective for detecting chromatin-binding proteins, such as SMARCC2, which are difficult to label using conventional IHC methods. Using CHIER on formalin-fixed paraffin-embedded human brain sections, we achieved robust detection of SMARCC2 in both the nucleus and cytoplasm. CHIER also enhanced the visualisation of large SMARCC2+ cytoplasmic bodies, termed cytobodies, which are increased in Parkinson’s Disease (PD). Our findings suggest that SMARCC2 may translocate from the nucleus to the cytoplasm in PD, potentially implicating SMARCC2 aggregation in the disease’s pathology. Furthermore, CHIER does not negatively impact the antigenicity of other antibodies, supporting its use for multiplex fluorescent immunohistochemistry and super-resolution imaging. These results highlight CHIER’s potential for improving the detection of chromatin-binding and aggregated proteins in neurodegenerative disease research, offering new insights into SMARCC2’s role in Parkinson’s Disease.

Mild traumatic brain injury (mTBI), often called concussion, is a prevalent condition that can have significant implications for people's health, functioning and well-being. Current clinical practice relies on self-reported symptoms to inform return to sport, work or school decisions, which can be highly problematic. An objective technique to detect the impact of mTBI on the brain is needed. MRI-based T2 relaxation is a quantitative imaging technique that is susceptible to detecting fluid properties in the brain and is a promising marker for detecting subtle neuroinflammation. This study aimed to investigate the potential of T2 relaxometry MRI in assessing mTBI at the individual level. The current study included 20 male participants with acute sports-related mTBI (within 14 days post-injury) and 44 healthy controls. We statistically compared each mTBI participant's voxel-wise T2 relaxometry map with the average of controls using a voxel-wise z-test with false discovery rate correction. In addition, five participants were re-scanned after clinical recovery, and their acute scans were compared to their recovery scans. Results revealed significantly increased T2 relaxation times in 19/20 (95%) of mTBI individuals, compared to controls, in multiple regions, including the hippocampus, frontal cortex, parietal cortex, insula, cingulate cortex and cerebellum. This suggests the presence of increased cerebral fluid in individuals with mTBI. Longitudinal results indicated a partial reduction in T2 relaxation for all five participants, suggesting a resolution over time. This research highlights the potential of T2 relaxometry MRI as a non-invasive method for assessing neuroinflammation in mTBI. Identifying and monitoring neuroinflammation could aid in predicting recovery and developing individualised treatment plans for individuals with mTBI. Future research would benefit from repeating all MRI scans at recovery to evaluate whether T2-relaxometry normalises or persists.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis study was funded by the Health Research Council of New Zealand (HRC)### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The Health and Disability Ethics Committee of New Zealand gave ethical approval for this work (HDEC 2022 EXP 11078)I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

BACKGROUND AND PURPOSE:The spinal cord is a key structure involved in the transmission and modulation of pain. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP), are expressed in the spinal cord. These peptides activate G protein-coupled receptors (PAC1, VPAC1 and VPAC2) that could provide targets for the development of novel pain treatments. However, it is not clear which of these receptors are expressed within the spinal cord and how these receptors signal. EXPERIMENTAL APPROACH:Dissociated rat spinal cord cultures were used to examine agonist and antagonist receptor pharmacology. Signalling profiles were determined for five signalling pathways. The expression of different PACAP and VIP receptors was then investigated in mouse, rat and human spinal cords using immunoblotting and immunofluorescence. KEY RESULTS:PACAP, but not VIP, potently stimulated cAMP, IP1 accumulation and ERK and cAMP response element-binding protein (CREB) but not Akt phosphorylation in spinal cord cultures. Signalling was antagonised by M65 and PACAP6-38. PACAP-27 was more effectively antagonised than either PACAP-38 or VIP. The patterns of PAC1 and VPAC2 receptor-like immunoreactivity appeared to be distinct in the spinal cord. CONCLUSIONS AND IMPLICATIONS:The pharmacological profile in the spinal cord suggested that a PAC1 receptor is the major functional receptor subtype present and thus likely mediates the nociceptive effects of the PACAP family of peptides in the spinal cord. However, the potential expression of both PAC1 and VPAC2 receptors in the spinal cord highlights that these receptors may play differential roles and are both possible therapeutic targets.