RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS.

TDP-43 is an aggregation-prone protein which accumulates in the hallmark pathological inclusions of amyotrophic lateral sclerosis (ALS). However, the analysis of deeply phenotyped human post-mortem samples has shown that TDP-43 aggregation, revealed by standard antibody methods, correlates poorly with symptom manifestation. Recent identification of cryptic-splicing events, such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism but the temporal nature of TDP-43 loss and its relation to the disease process and clinical phenotype is not known. To address these outstanding questions, we used a novel RNA aptamer, TDP-43APT, to detect TDP-43 pathology and used single molecule in situ hybridization to sensitively reveal TDP-43 loss-of-function and applied these in a deeply phenotyped human post-mortem tissue cohort. We demonstrate that TDP-43APT identifies pathological TDP-43, detecting aggregation events that cannot be detected by classical antibody stains. We show that nuclear TDP-43 pathology is an early event, occurring prior to cytoplasmic accumulation and is associated with loss-of-function measured by coincident STMN-2 cryptic splicing pathology. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function in the form of extensive cytoplasmic accumulation, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics as the presence of STMN-2 cryptic exons and early TDP-43 aggregation events could be detected prior to symptom onset, holding promise for early intervention in ALS.

Distinct neuroinflammatory signatures exist across genetic and sporadic amyotrophic lateral sclerosis cohorts.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. ALS is on a pathogenetic disease spectrum with frontotemporal dementia, referred to as ALS-frontotemporal spectrum disorder (ALS-FTSD). For mutations associated with ALS-FTSD, such as the C9orf72 hexanucleotide repeat expansion, the molecular factors associated with heterogeneity along this spectrum require further characterization. Here, using a targeted NanoString molecular barcoding approach, we interrogate neuroinflammatory dysregulation and heterogeneity at the level of gene expression in post-mortem motor cortex tissue from a cohort of clinically heterogeneous C9-ALS-FTSD cases. We identified 20 dysregulated genes in C9-ALS-FTSD, with enrichment of microglial and inflammatory response gene sets. Two genes with significant correlations to available clinical metrics were selected for validation: FKBP5, a correlate of cognitive function, and brain-derived neurotrophic factor (BDNF), a correlate of disease duration. FKBP5 and its signalling partner, NF-κB, appeared to have a cell type-specific staining distribution, with activated (i.e. nuclear) NF-κB immunoreactivity in C9-ALS-FTSD. Expression of BDNF, a correlate of disease duration, was confirmed to be higher in individuals with long compared to short disease duration using BaseScope™ in situ hybridization. Our analyses also revealed two distinct neuroinflammatory panel signatures (NPS), NPS1 and NPS2, delineated by the direction of expression of proinflammatory, axonal transport and synaptic signalling pathways. We compared NPS between C9-ALS-FTSD cases and those from sporadic ALS and SOD1-ALS cohorts and identified NPS1 and NPS2 across all cohorts. Moreover, a subset of NPS was also able to separate publicly available RNA sequencing data from independent C9-ALS and sporadic ALS cohorts into two inflammatory subgroups. Importantly, NPS subgroups did not clearly segregate with available demographic, genetic, clinical or pathological features, highlighting the value of molecular stratification in clinical trials for inflammatory subgroup identification. Our findings thus underscore the importance of tailoring therapeutic approaches based on distinct molecular signatures that exist between and within ALS-FTSD cohorts.

pTDP-43 aggregates accumulate in non-central nervous system tissues prior to symptom onset in amyotrophic lateral sclerosis: a case series linking archival surgical biopsies with clinical phenotypic data.

Neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) are traditionally considered strictly neurological disorders. However, clinical presentation is not restricted to neurological systems, and non-central nervous system (CNS) manifestations, particularly gastrointestinal (GI) symptoms, are common. Our objective was to understand the systemic distribution of pathology in archived non-CNS tissues, taken as part of routine clinical practice during life from people with ALS. We examined tissue from 13 people who went on to develop ALS; including sporadic ALS (n = 12) and C9orf72 hexanucleotide repeat expansion (n = 1). The tissue cohort consisted of 68 formalin-fixed paraffin embedded samples from 21 surgical cases (some patients having more than one case over their lifetimes), from 8 organ systems, which we examined for evidence of phosphorylated TDP-43 (pTDP-43) pathology. We identified pTDP-43 aggregates in multiple cell types of the GI tract, including macrophages and dendritic cells within the lamina propria; as well as ganglion/neuronal and glial cells of the myenteric plexus. Aggregates were also noted within lymph node parenchyma, blood vessel endothelial cells, and chondrocytes. We note that in all cases with non-CNS pTDP-43 pathology, aggregates were present prior to ALS diagnosis and in some instances preceded neurological symptom onset by more than 10 years. These data imply that patients with microscopically unexplained non-CNS symptoms could have occult protein aggregation that could be detected many years prior to neurological involvement.

Random forest modelling demonstrates microglial and protein misfolding features to be key phenotypic markers in C9orf72-ALS.

Clinical heterogeneity observed across patients with amyotrophic lateral sclerosis (ALS) is a known complicating factor in identifying potential therapeutics, even within cohorts with the same mutation, such as C9orf72 hexanucleotide repeat expansions (HREs). Thus, further understanding of pathways underlying this heterogeneity is essential for appropriate ALS trial stratification and the meaningful assessment of clinical outcomes. It has been shown that both inflammation and protein misfolding can influence ALS pathogenesis, such as the manifestation or severity of motor or cognitive symptoms. However, there has yet to be a systematic and quantitative assessment of immunohistochemical markers to interrogate the potential relevance of these pathways in an unbiased manner. To investigate this, we extensively characterised features of commonly used glial activation and protein misfolding stains in thousands of images of post-mortem tissue from a heterogeneous cohort of deeply clinically profiled patients with a C9orf72 HRE. Using a random forest model, we show that microglial staining features are the most accurate classifiers of disease status in our panel and that clinicopathological relationships exist between microglial activation status, TDP-43 pathology, and language dysfunction. Furthermore, we detected spatially resolved changes in fused in sarcoma (FUS) staining, suggesting that liquid-liquid phase shift of this aggregation-prone RNA-binding protein may be important in ALS caused by a C9orf72 HRE. Interestingly, no one feature alone significantly impacted the predictiveness of the model, indicating that the collective examination of all features, or a combination of several features, is what allows the model to be predictive. Our findings provide further support to the hypothesis of dysfunctional immune regulation and proteostasis in the pathogenesis of C9-ALS and provide a framework for digital analysis of commonly used neuropathological stains as a tool to enrich our understanding of clinicopathological relationships within and between cohorts. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Investigating Markers of Rapport in Autistic and Nonautistic Interactions.

Background: Autism is considered to entail a social impairment whereby autistic people experience difficulty interpreting others' mental states. However, recent research has shown that nonautistic people also have difficulty understanding the mental states of autistic people. This mismatch of understanding may explain lower rapport in interactions between autistic and nonautistic people. As mental states can be expressed externally through socially normed signals, it is important to investigate the role of such signals in autistic, nonautistic, and mixed interactions. This study explores variability in two social signals between autistic, nonautistic, and mixed interactions, and how their use may affect rapport within interactions. Methods: Videos from a previous study of autistic, nonautistic, and mixed pair interactions in a diffusion chain context in which participants were aware of others' diagnostic status were video coded for mutual gaze and backchanneling as candidate indicators of interactional rapport. Results: Although use of mutual gaze and backchanneling was lower in mixed pairs than in nonautistic pairs, corresponding to lower ratings of interactional rapport, less backchanneling in autistic pairs of both nonverbal and verbal subtypes corresponded to higher ratings of rapport. Conclusions: We observed differences in the use of candidate rapport markers between autistic, mixed, and nonautistic interactions, which did not map onto patterns of rapport scores, suggesting differences in reliance on these cues between autistic and nonautistic people. These results suggest that visible markers of rapport may vary by neurotype or pairing and give clues to inform future investigations of autistic interaction.

Why is this an important issue?: When someone is autistic, society generally assumes they have difficulty interacting with others. Social difficulties between autistic and nonautistic people are thought to be due to the autistic person not being able to interact using nonautistic social behaviors. This belief can lead to many autistic people feeling alienated. However, recent research supports what autistic people have been saying for a long time: that autistic people are capable of having successful and rewarding interactions with other autistic people. This suggests that social difficulties between autistic and nonautistic people may be due to a mismatch in social norms leading to difficulty for both people, not just the autistic person. What was the purpose of this study?: In this study, we wanted to investigate whether certain social signals are used differently between autistic and nonautistic people. We also questioned whether using these signals helped or hurt the interaction depending on who was involved. What did the researchers do?: We recorded people passing a story down a chain of people, like the game telephone, to see how they interacted with each other. Afterward, people wrote down scores for how much they enjoyed their interaction. We focused on two parts of the interaction: how much people were looking at each other and when they made short verbal responses such as "mhm" to show they were listening (backchanneling). We watched the recordings back and analyzed how long or how many times these actions were occurring. What were the results of the study?: We found that when one autistic and one nonautistic person were interacting, they looked at each other and backchanneled less than two nonautistic people. This seemed to be linked with a less enjoyable interaction for them. However, backchanneling seemed to matter less in interactions between two autistic people. They backchanneled less while still having enjoyable interactions. What do these findings add to what was already known?: Research has previously suggested that different social norms exist between autistic and nonautistic people. This study shows specific examples of this and how they may affect the interaction in a natural setting. What are potential weaknesses in the study?: This study has some weaknesses. For example, we measured when people looked at each other's faces rather than eye contact specifically, which can only be done with an eye tracker. Also, the people in the study knew whether the person they were talking to was autistic. This can be similar to normal life, as people do sometimes know this, although we would also be interested to see what the effect of not knowing would be. How will these findings help autistic adults now or in the future?: This line of research has important implications for how autistic people can be supported in society. Not only will understanding of social differences between autistic and nonautistic people help the way autistic people are perceived, but it will also help nonautistic people better understand and support the autistic people in their lives.

NLRP3 inflammasome as a key molecular target underlying cognitive resilience in amyotrophic lateral sclerosis.

Up to 50% of amyotrophic lateral sclerosis patients present with cognitive deficits in addition to motor dysfunction, but the molecular mechanisms underlying diverse clinical and pathological presentations remain poorly understood. There is therefore an unmet need to identify molecular drivers of cognitive dysfunction to enable better therapeutic targeting and prognostication. To address this, we employed a non-biased approach to identify molecular targets using a deeply phenotyped, clinically stratified cohort of cognitively affected and unaffected brain regions from three brain regions of 13 amyotrophic lateral sclerosis patients with the same cognitive screening test performed during life. Using NanoString molecular barcoding as a sensitive mRNA sequencing technique on post-mortem tissue, we profiled a data-driven panel of 770 genes using the Neuropathology Panel, followed by region and cell type-specific validation using BaseScope in situ hybridisation and immunohistochemistry. We identified 50 significantly dysregulated genes that are distinct between cognitively affected and unaffected brain regions. Using BaseScope in situ hybridisation, we also demonstrate that macromolecular complex regulation, notably NLRP3 inflammasome modulation, is a potential, therapeutically targetable, pathological correlate of cognitive resilience in ALS. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Associations between major psychiatric disorder polygenic risk scores and blood-based markers in UK biobank.

Major depressive disorder (MDD), schizophrenia (SCZ), and bipolar disorder (BD) have both shared and discrete genetic risk factors, and are associated with peripheral abnormalities. The relationships between such genetic architectures and blood-based markers are, however, unclear. We investigated relationships between polygenic risk scores (PRS) for these disorders and peripheral markers in the UK Biobank cohort. We calculated polygenic risk scores for n = 367,329 (MDD PRS), n = 366,465 (SCZ PRS), and n = 366,383 (BD PRS) UK Biobank cohort subjects. We then examined associations between disorder PRS and 58 inflammatory/immune, hematological, bone, cardiovascular, hormone, liver, renal and diabetes-associated blood markers using two generalized linear regression models: 'minimally adjusted' controlling for variables such as age and sex, and 'fully adjusted' including additional lifestyle covariates: BMI, alcohol and smoking status, and medication intake. There were 38/58 MDD PRS, 32/58 SCZ PRS, and 20/58 BD PRS-blood marker associations detected for our minimally adjusted model. Of these, 13/38 (MDD PRS), 14/32 (SCZ PRS), and 10/20 (BD PRS) associations remained significant after controlling for lifestyle factors. Many were disorder-specific, with 8/13 unique MDD PRS associations identified. Several disorder-specific associations for MDD and SCZ were immune-related, with mostly positive and negative associations identified for MDD and SCZ PRS respectively. This study suggests that MDD, SCZ and BD have both shared and distinct peripheral markers associated with disorder-specific genetic risk. The results also implicate inflammatory dysfunction in MDD and SCZ, albeit with differences in patterns between the two conditions, and enrich our understanding of potential underlying pathophysiological mechanisms in major psychiatric disorders.

Toxicity of pathogenic ataxin-2 in Drosophila shows dependence on a pure CAG repeat sequence.

Spinocerebellar ataxia type 2 is a polyglutamine (polyQ) disease associated with an expanded polyQ domain within the protein product of the ATXN2 gene. Interestingly, polyQ repeat expansions in ATXN2 are also associated with amyotrophic lateral sclerosis (ALS) and parkinsonism depending upon the length of the polyQ repeat expansion. The sequence encoding the polyQ repeat also varies with disease presentation: a pure CAG repeat is associated with SCA2, whereas the CAG repeat in ALS and parkinsonism is typically interrupted with the glutamine encoding CAA codon. Here, we asked if the purity of the CAG sequence encoding the polyQ repeat in ATXN2 could impact the toxicity of the ataxin-2 protein in vivo in Drosophila. We found that ataxin-2 encoded by a pure CAG repeat conferred toxicity in the retina and nervous system, whereas ataxin-2 encoded by a CAA-interrupted repeat or CAA-only repeat failed to confer toxicity, despite expression of the protein at similar levels. Furthermore, the CAG-encoded ataxin-2 protein aggregated in the fly eye, while ataxin-2 encoded by either a CAA/G or CAA repeat remained diffuse. The toxicity of the CAG-encoded ataxin-2 protein was also sensitive to the translation factor eIF4H, a known modifier of the toxic GGGGCC repeat in flies. These data indicate that ataxin-2 encoded by a pure CAG versus interrupted CAA/G polyQ repeat domain is associated with differential toxicity, indicating that mechanisms associated with the purity of the sequence of the polyQ domain contribute to disease.

The application of optical coherence tomography angiography in Alzheimer's disease: A systematic review.

INTRODUCTION: Discovering non-invasive and easily acquired biomarkers that are conducive to the accurate diagnosis of dementia is an urgent area of ongoing clinical research. One promising approach is retinal imaging, as there is homology between retinal and cerebral vasculature. Recently, optical coherence tomography angiography (OCT-A) has emerged as a promising new technology for imaging the microvasculature of the retina. METHODS: A systematic review and meta-analysis was conducted to examine the application of OCT-A in dementia. RESULTS: Fourteen studies assessing OCT-A in preclinical Alzheimer's disease (AD), mild cognitive impairment, or AD were included. Exploratory meta-analyses revealed a significant increase in the foveal avascular zone area and a significant decrease in superficial parafoveal and whole vessel density in AD, although there was significant heterogeneity between studies. DISCUSSION: Although certain OCT-A metrics may have the potential to serve as biomarkers for AD, the field requires further standardization to allow conclusions to be reached regarding their clinical utility.

TDP-43, a protein central to amyotrophic lateral sclerosis, is destabilized by tankyrase-1 and -2.

In >95% of cases of amyotrophic lateral sclerosis (ALS) and ∼45% of frontotemporal degeneration (FTD), the RNA/DNA-binding protein TDP-43 is cleared from the nucleus and abnormally accumulates in the cytoplasm of affected brain cells. Although the cellular triggers of disease pathology remain enigmatic, mounting evidence implicates the poly(ADP-ribose) polymerases (PARPs) in TDP-43 neurotoxicity. Here we show that inhibition of the PARP enzymes tankyrase 1 and tankyrase 2 (referred to as Tnks-1/2) protect primary rodent neurons from TDP-43-associated neurotoxicity. We demonstrate that Tnks-1/2 interacts with TDP-43 via a newly defined tankyrase-binding domain. Upon investigating the functional effect, we find that interaction with Tnks-1/2 inhibits the ubiquitination and proteasomal turnover of TDP-43, leading to its stabilization. We further show that proteasomal turnover of TDP-43 occurs preferentially in the nucleus; our data indicate that Tnks-1/2 stabilizes TDP-43 by promoting cytoplasmic accumulation, which sequesters the protein from nuclear proteasome degradation. Thus, Tnks-1/2 activity modulates TDP-43 and is a potential therapeutic target in diseases associated with TDP-43, such as ALS and FTD.This article has an associated First Person interview with the first author of the paper.

Poly(ADP-Ribosylation) in Age-Related Neurological Disease.

A central and causative feature of age-related neurodegenerative disease is the deposition of misfolded proteins in the brain. To devise novel approaches to treatment, regulatory pathways that modulate these aggregation-prone proteins must be defined. One such pathway is post-translational modification by the addition of poly(ADP-ribose) (PAR), which promotes protein recruitment and localization in several cellular contexts. Mounting evidence implicates PAR in seeding the abnormal localization and accumulation of proteins that are causative of neurodegenerative disease. Inhibitors of PAR polymerase (PARP) activity have been developed as cancer therapeutics, raising the possibility that they could be used to treat neurodegenerative disease. We focus on pathways regulated by PAR in neurodegenerative disease, with emphasis on amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD).

eIF4B and eIF4H mediate GR production from expanded G4C2 in a Drosophila model for C9orf72-associated ALS.

The discovery of an expanded (GGGGCC)n repeat (termed G4C2) within the first intron of C9orf72 in familial ALS/FTD has led to a number of studies showing that the aberrant expression of G4C2 RNA can produce toxic dipeptides through repeat-associated non-AUG (RAN-) translation. To reveal canonical translation factors that impact this process, an unbiased loss-of-function screen was performed in a G4C2 fly model that maintained the upstream intronic sequence of the human gene and contained a GFP tag in the GR reading frame. 11 of 48 translation factors were identified that impact production of the GR-GFP protein. Further investigations into two of these, eIF4B and eIF4H, revealed that downregulation of these factors reduced toxicity caused by the expression of expanded G4C2 and reduced production of toxic GR dipeptides from G4C2 transcripts. In patient-derived cells and in post-mortem tissue from ALS/FTD patients, eIF4H was found to be downregulated in cases harboring the G4C2 mutation compared to patients lacking the mutation and healthy individuals. Overall, these data define eIF4B and eIF4H as disease modifiers whose activity is important for RAN-translation of the GR peptide from G4C2-transcripts.