Apilimod dimesylate in C9orf72 amyotrophic lateral sclerosis: a randomized phase 2a clinical trial.

Apilimod dimesylate is a first-in-class phosphoinositide kinase, FYVE-type zinc finger containing (PIKfyve) inhibitor with favourable clinical safety profile and has demonstrated activity in preclinical C9orf72 and TDP-43 amyotrophic lateral sclerosis models. In this amyotrophic lateral sclerosis clinical trial, the safety, tolerability, CNS penetrance, and modulation of pharmacodynamic target engagement biomarkers were evaluated. This Phase 2a, randomized, double-blind, placebo-controlled, biomarker-endpoint clinical trial was conducted in four USA centres (ClinicalTrials.gov NCT05163886). Participants with C9orf72 repeat expansion were randomly assigned (2:1) to receive twice-daily oral treatment of 125 mg apilimod dimesylate capsules or matching placebo for 12 weeks, followed by a 12-week open-label extension. Safety was measured as the occurrence of treatment-emergent adverse or serious adverse events attributable to study drug, and tolerability as trial completion on treatment over 12 weeks. Changes from baseline in plasma and CSF and concentrations of apilimod and its active metabolites and of pharmacodynamic biomarkers of PIKfyve inhibition (soluble glycoprotein nonmetastatic melanoma protein B [sGPNMB] upregulation) and disease-specific CNS target engagement (poly[GP]). Between Dec 16, 2021, and Jul 7, 2022, 15 eligible participants were enrolled. There were no drug-related serious adverse events reported in the trial. Fourteen (93%) participants completed the double-blind period with 99% dose compliance (N=9 [90%] apilimod dimesylate; N=5 [100%] placebo). At Week 12, apilimod dimesylate was measurable in CSF at 1.63 ng/mL (SD: 0.937). At Week 12, apilimod dimesylate increased plasma sGPNMB by > 2.5-fold (p < 0.001) indicating PIKfyve inhibition and lowered CSF poly(GP) protein levels by 73% (p < 0.001) indicating CNS tissue-level proof of mechanism. Apilimod dimesylate met prespecified key safety and biomarker endpoints in this Phase 2a trial and demonstrated CNS penetrance and pharmacodynamic target engagement. Apilimod dimesylate was observed to have the greatest reduction in CSF poly(GP) levels observed to date in C9orf72 clinical trials.

Generation and characterization of monoclonal antibodies against pathologically phosphorylated TDP-43.

Inclusions containing TAR DNA binding protein 43 (TDP-43) are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). One of the disease-specific features of TDP-43 inclusions is the aberrant phosphorylation of TDP-43 at serines 409/410 (pS409/410). Here, we developed rabbit monoclonal antibodies (mAbs) that specifically detect pS409/410-TDP-43 in multiple model systems and FTD/ALS patient samples. Specifically, we identified three mAbs (26H10, 2E9 and 23A1) from spleen B cell clones that exhibit high specificity and sensitivity to pS409/410-TDP-43 peptides in an ELISA assay. Biochemical analyses revealed that pS409/410 of recombinant TDP-43 and of exogenous 25 kDa TDP-43 C-terminal fragments in cultured HEK293T cells are detected by all three mAbs. Moreover, the mAbs detect pS409/410-positive TDP-43 inclusions in the brains of FTD/ALS patients and mouse models of TDP-43 proteinopathy by immunohistochemistry. Our findings indicate that these mAbs are a valuable resource for investigating TDP-43 pathology both in vitro and in vivo.

HDGFL2 cryptic proteins report presence of TDP-43 pathology in neurodegenerative diseases.

This letter demonstrates the potential of novel cryptic proteins resulting from TAR DNA-binding protein 43 (TDP-43) dysfunction as markers of TDP-43 pathology in neurodegenerative diseases.

Erratum: Neurofilament light chain and vaccination status associate with clinical outcomes in severe COVID-19.

[This corrects the article DOI: 10.1016/j.isci.2022.105272.].

TDP-43 nuclear loss in FTD/ALS causes widespread alternative polyadenylation changes.

In frontotemporal dementia and amyotrophic lateral sclerosis, the RNA-binding protein TDP-43 is depleted from the nucleus. TDP-43 loss leads to cryptic exon inclusion but a role in other RNA processing events remains unresolved. Here, we show that loss of TDP-43 causes widespread changes in alternative polyadenylation, impacting expression of disease-relevant genes (e.g., ELP1, NEFL, and TMEM106B) and providing evidence that alternative polyadenylation is a new facet of TDP-43 pathology.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

TMEM106B core deposition associates with TDP-43 pathology and is increased in risk SNP carriers for frontotemporal dementia.

Genetic variation at the transmembrane protein 106B gene (TMEM106B) has been linked to risk of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) through an unknown mechanism. We found that presence of the TMEM106B rs3173615 protective genotype was associated with longer survival after symptom onset in a postmortem FTLD-TDP cohort, suggesting a slower disease course. The seminal discovery that filaments derived from TMEM106B is a common feature in aging and, across a range of neurodegenerative disorders, suggests that genetic variants in TMEM106B could modulate disease risk and progression through modulating TMEM106B aggregation. To explore this possibility and assess the pathological relevance of TMEM106B accumulation, we generated a new antibody targeting the TMEM106B filament core sequence. Analysis of postmortem samples revealed that the TMEM106B rs3173615 risk allele was associated with higher TMEM106B core accumulation in patients with FTLD-TDP. In contrast, minimal TMEM106B core deposition was detected in carriers of the protective allele. Although the abundance of monomeric full-length TMEM106B was unchanged, carriers of the protective genotype exhibited an increase in dimeric full-length TMEM106B. Increased TMEM106B core deposition was also associated with enhanced TDP-43 dysfunction, and interactome data suggested a role for TMEM106B core filaments in impaired RNA transport, local translation, and endolysosomal function in FTLD-TDP. Overall, these findings suggest that prevention of TMEM106B core accumulation is central to the mechanism by which the TMEM106B protective haplotype reduces disease risk and slows progression.

CRISPR interference to evaluate modifiers of C9ORF72-mediated toxicity in FTD.

Treatments for neurodegenerative disease, including Frontotemporal dementia (FTD) and Amyotrophic lateral sclerosis (ALS), remain rather limited, underscoring the need for greater mechanistic insight and disease-relevant models. Our ability to develop novel disease models of genetic risk factors, disease modifiers, and other FTD/ALS-relevant targets is impeded by the significant amount of time and capital required to develop conventional knockout and transgenic mice. To overcome these limitations, we have generated a novel CRISPRi interference (CRISPRi) knockin mouse. CRISPRi uses a catalytically dead form of Cas9, fused to a transcriptional repressor to knockdown protein expression, following the introduction of single guide RNA against the gene of interest. To validate the utility of this model we have selected the TAR DNA binding protein (TDP-43) splicing target, stathmin-2 (STMN2). STMN2 RNA is downregulated in FTD/ALS due to loss of TDP-43 activity and STMN2 loss is suggested to play a role in ALS pathogenesis. The involvement of STMN2 loss of function in FTD has yet to be determined. We find that STMN2 protein levels in familial FTD cases are significantly reduced compared to controls, supporting that STMN2 depletion may be involved in the pathogenesis of FTD. Here, we provide proof-of-concept that we can simultaneously knock down Stmn2 and express the expanded repeat in the Chromosome 9 open reading frame 72 (C9ORF72) gene, successfully replicating features of C9-associated pathology. Of interest, depletion of Stmn2 had no effect on expression or deposition of dipeptide repeat proteins (DPRs), but significantly decreased the number of phosphorylated Tdp-43 (pTdp-43) inclusions. We submit that our novel CRISPRi mouse provides a versatile and rapid method to silence gene expression in vivo and propose this model will be useful to understand gene function in isolation or in the context of other neurodegenerative disease models.

TDP-43-regulated cryptic RNAs accumulate in Alzheimer's disease brains.

BACKGROUND: Inclusions of TAR DNA-binding protein 43 kDa (TDP-43) has been designated limbic-predominant, age-related TDP-43 encephalopathy (LATE), with or without co-occurrence of Alzheimer's disease (AD). Approximately, 30-70% AD cases present TDP-43 proteinopathy (AD-TDP), and a greater disease severity compared to AD patients without TDP-43 pathology. However, it remains unclear to what extent TDP-43 dysfunction is involved in AD pathogenesis. METHODS: To investigate whether TDP-43 dysfunction is a prominent feature in AD-TDP cases, we evaluated whether non-conserved cryptic exons, which serve as a marker of TDP-43 dysfunction in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), accumulate in AD-TDP brains. We assessed a cohort of 192 post-mortem brains from three different brain regions: amygdala, hippocampus, and frontal cortex. Following RNA and protein extraction, qRT-PCR and immunoassays were performed to quantify the accumulation of cryptic RNA targets and phosphorylated TDP-43 pathology, respectively. RESULTS: We detected the accumulation of misspliced cryptic or skiptic RNAs of STMN2, KCNQ2, UNC13A, CAMK2B, and SYT7 in the amygdala and hippocampus of AD-TDP cases. The topographic distribution of cryptic RNA accumulation mimicked that of phosphorylated TDP-43, regardless of TDP-43 subtype classification. Further, cryptic RNAs efficiently discriminated AD-TDP cases from controls. CONCLUSIONS: Overall, our results indicate that cryptic RNAs may represent an intriguing new therapeutic and diagnostic target in AD, and that methods aimed at detecting and measuring these species in patient biofluids could be used as a reliable tool to assess TDP-43 pathology in AD. Our work also raises the possibility that TDP-43 dysfunction and related changes in cryptic splicing could represent a common molecular mechanism shared between AD-TDP and FTLD-TDP.

Correction: TDP-43 and other hnRNPs regulate cryptic exon inclusion of a key ALS/FTD risk gene, UNC13A.

[This corrects the article DOI: 10.1371/journal.pbio.3002028.].

Perry syndrome: Novel DCTN1 mutation in a large kindred and first observation of prodromal disease.

INTRODUCTION: Perry syndrome (PS) is a hereditary neurodegenerative disorder caused by mutations in the DCTN1 gene and characterized by TDP-43 pathology. As the diagnosis is usually made at the advanced stages of the disease, there are no studies on the asymptomatic mutation carriers and their conversion to overt disease. METHODS: We personally examined 27 members of the large kindred of 104 individuals with familial parkinsonism. We evaluated each case with clinical (neurological examination; motor and non-motor scales), genetic testing (whole-exome or Sanger sequencing), and laboratory (neurofilament light, NFL; glial fibrillary acidic protein, GFAP) measures. Autopsy study was done on two individuals. RESULTS: The mean age at evaluation was 49 years. Comorbidities were present in 20 cases, including sleep problems (n = 15 total, sleep apnea in 7), dysautonomia (n = 10), weight loss (n = 8), and anxiety/depression (n = 8). Neurological abnormalities were present in 18, including parkinsonism (n = 7), isolated tremor (n = 2), and varied isolated signs in individual cases. Cognition and smell were preserved. Genetic testing revealed a novel c.200G > T (Gly67Val) mutation in the DCTN1 gene in 10 individuals. The mutation, segregated with the PS phenotype (n = 4), was absent in gnomAD, and in silico predictions indicated it was pathogenic. Three young mutation carriers were monosymptomatic (prodromal), and three were asymptomatic. Plasma NFL and GFAP values were similar among the cases. Autopsy studies showed typical PS neuropathological findings. CONCLUSIONS: We identified a novel pathogenic Gly67Val DCTN1 mutation. We report prodromal disease of PS in some mutation carriers; however, more investigation is necessary to confirm this observation.

First families with spinocerebellar ataxia type 7 in Poland.

INTRODUCTION: We present the first two Polish families diagnosed with spinocerebellar ataxia type 7 (SCA7) and draw attention to cardiac involvement as a new potential manifestation of this disease. MATERIAL AND METHODS: Two well-documented kindreds are presented. RESULTS: The proband from Family 1 presented aged 54 years with vision worsening followed by progressive imbalance. Brain MRI demonstrated cerebellar atrophy. Genetic testing confirmed CAG repeat expansion (42/10) in ATXN7 gene. The proband from Family 2 developed imbalance at age 20, followed by progressive deterioration of vision. Brain MRI revealed cerebellar atrophy. Additionally, she developed chronic congestive heart failure and, at age 38, had cardiomyopathy with an ejection fraction of 20% and significant mitral and tricuspid regurgitation. Genetic analysis found abnormal CAG expansion in the ATXN7 (46/10). CONCLUSIONS AND CLINICAL IMPLICATIONS: Vision loss due to pigmentary retinal degeneration is the distinguishing feature of SCA7 and often the initial manifestation. Although SCA7 is one of the most common SCAs in Sweden, it has never been reported in neighbouring Poland. Until now, cardiac abnormalities have only been described in infantile-onset SCA7 with large CAG repeats. The observed cardiac involvement in Family 2 may be coincidental, albeit a new possible manifestation of SCA7 cannot be excluded.

Prognostic performance of blood neurofilament light chain protein in hospitalized COVID-19 patients without major central nervous system manifestations: an individual participant data meta-analysis.

BACKGROUND AND AIMS: To investigate the prognostic value of blood neurofilament light chain protein (NfL) levels in the acute phase of coronavirus disease 2019 (COVID-19). METHODS: We conducted an individual participant data (IPD) meta-analysis after screening on MEDLINE and Scopus to May 23rd 2022. We included studies with hospitalized adult COVID-19 patients without major COVID-19-associated central nervous system (CNS) manifestations and with a measurement of blood NfL in the acute phase as well as data regarding at least one clinical outcome including intensive care unit (ICU) admission, need of mechanical ventilation (MV) and death. We derived the age-adjusted measures NfL Z scores and conducted mixed-effects modelling to test associations between NfL Z scores and other variables, encompassing clinical outcomes. Summary receiver operating characteristic curves (SROCs) were used to calculate the area under the curve (AUC) for blood NfL. RESULTS: We identified 382 records, of which 7 studies were included with a total of 669 hospitalized COVID-19 cases (mean age 66.2 ± 15.0 years, 68.1% males). Median NfL Z score at admission was elevated compared to the age-corrected reference population (2.37, IQR: 1.13-3.06, referring to 99th percentile in healthy controls). NfL Z scores were significantly associated with disease duration and severity. Higher NfL Z scores were associated with a higher likelihood of ICU admission, need of MV, and death. SROCs revealed AUCs of 0.74, 0.80 and 0.71 for mortality, need of MV and ICU admission, respectively. CONCLUSIONS: Blood NfL levels were elevated in the acute phase of COVID-19 patients without major CNS manifestations and associated with clinical severity and poor outcome. The marker might ameliorate the performance of prognostic multivariable algorithms in COVID-19.

TDP-43 and other hnRNPs regulate cryptic exon inclusion of a key ALS/FTD risk gene, UNC13A.

A major function of TAR DNA-binding protein-43 (TDP-43) is to repress the inclusion of cryptic exons during RNA splicing. One of these cryptic exons is in UNC13A, a genetic risk factor for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The accumulation of cryptic UNC13A in disease is heightened by the presence of a risk haplotype located within the cryptic exon itself. Here, we revealed that TDP-43 extreme N-terminus is important to repress UNC13A cryptic exon inclusion. Further, we found hnRNP L, hnRNP A1, and hnRNP A2B1 bind UNC13A RNA and repress cryptic exon inclusion, independently of TDP-43. Finally, higher levels of hnRNP L protein associate with lower burden of UNC13A cryptic RNA in ALS/FTD brains. Our findings suggest that while TDP-43 is the main repressor of UNC13A cryptic exon inclusion, other hnRNPs contribute to its regulation and may potentially function as disease modifiers.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.

Race affects adverse outcomes of deep vein thrombosis, pulmonary embolism, and acute kidney injury in coronavirus disease 2019 hospitalized patients.

OBJECTIVE: The purpose of the present study was to explore the racial disparities in the incidence of deep vein thrombosis (DVT), pulmonary embolism (PE), and acute kidney injury (AKI) in hospitalized patients with coronavirus disease 2019 (COVID-19). METHODS: A retrospective analysis was performed of prospectively collected data of consecutive COVID-19 patients hospitalized from March 11, 2020 to May 27, 2021. The primary outcome measures were the incidence of DVT/PE and mortality. The secondary outcome measures included differences in the length of hospitalization, need for intensive care unit care, readmission, and AKI. Multivariable regression models were used to assess for independent predictors of the primary and secondary outcome measures. RESULTS: The present study included 876 hospitalized patients with COVID-19. The mean age was 64.4 ± 16.2 years, and 355 were women (40.5%). Of the 876 patients, 694 (79.2%) had identified as White, 111 (12.7%) as Black/African American, 48 (5.5%) as Asian, and 23 (2.6%) as other. The overall incidence of DVT/PE was 8.7%. The DVT/PE incidence rates differed across the race groups and was highest for Black/African American patients (n = 18; 16.2%), followed by Asian patients (n = 5; 10.4%), White patients (n = 52; 7.5%), and other (n = 1; 4.4%; P = .03). All but one of the hospitalization outcomes examined demonstrated no differences according to race, including the hospitalization stay (P = .33), need for intensive care unit care (P = .20), readmission rates (P = .52), and hospital all-cause mortality (P = .29). The AKI incidence differed among races, affecting a higher proportion of Black/African American patients (P=.003). On multivariable regression analysis, Black/African American race (odds ratio [OR], 2.0; 95% confidence interval [CI], 1.0-4.0; P = .04) and higher D-dimer levels (OR, 1.1; 95% CI, 1.1-1.2; P < .0001) were predictors of DVT/PE. In addition, Black/African American race (OR, 2.3; 95% CI, 1.4-3.7; P = .001), lower hemoglobin levels (OR, 0.84; 95% CI, 0.8-0.9; P ≤ .0001), male sex (OR, 1.7; 95% CI, 1.2-2.4; P = .005), hypertension (OR, 2.1; 95% CI, 1.4-3.1; P = .0005), and older age (OR, 1.02; 95% CI, 1.006-1.03; P = .003) were predictors of AKI. CONCLUSIONS: In our single-center case series, we found a higher incidence of DVT/PE and AKI among Black/African American patients with COVID-19. Black/African American race and D-dimer levels were independent predictors of DVT/PE, and Black/African American race, hemoglobin, and D-dimer levels were independent predictors of AKI.

ProtPipe: A Multifunctional Data Analysis Pipeline for Proteomics and Peptidomics.

Mass spectrometry (MS) is a technique widely employed for the identification and characterization of proteins, personalized medicine, systems biology and biomedical applications. By combining MS with different proteomics approaches such as immunopurification MS, immunopeptidomics, and total protein proteomics, researchers can gain insights into protein-protein interactions, immune responses, cellular processes, and disease mechanisms. The application of MS-based proteomics in these areas continues to advance our understanding of protein function, cellular signaling, and complex biological systems. Data analysis for mass spectrometry is a critical process that includes identifying and quantifying proteins and peptides and exploring biological functions for these proteins in downstream analysis. To address the complexities associated with MS data analysis, we developed ProtPipe to streamline and automate the processing and analysis of high-throughput proteomics and peptidomics datasets. The pipeline facilitates data quality control, sample filtering, and normalization, ensuring robust and reliable downstream analysis. ProtPipe provides downstream analysis including identifying differential abundance proteins and peptides, pathway enrichment analysis, protein-protein interaction analysis, and MHC1-peptide binding affinity. ProtPipe generates annotated tables and diagnostic visualizations from statistical postprocessing and computation of fold-changes across pairwise conditions, predefined in an experimental design. ProtPipe is well-documented open-source software and is available at https://github.com/NIH-CARD/ProtPipe , accompanied by a web interface.

Neurofilament light chain and vaccination status associate with clinical outcomes in severe COVID-19.

Blood neurofilament light chain (NFL) is proposed to serve as an estimate of disease severity in hospitalized patients with coronavirus disease 2019 (COVID-19). We show that NFL concentrations in plasma collected from 880 patients with COVID-19 within 5 days of hospital admission were elevated compared to controls. Higher plasma NFL associated with worse clinical outcomes including the need for mechanical ventilation, intensive care, prolonged hospitalization, and greater functional disability at discharge. No difference in the studied clinical outcomes between black/African American and white patients was found. Finally, vaccination associated with less disability at time of hospital discharge. In aggregate, our findings support the utility of measuring NFL shortly after hospital admission to estimate disease severity and show that race does not influence clinical outcomes caused by COVID-19 assuming equivalent access to care, and that vaccination may lessen the degree of COVID-19-caused disability.

Two FTD-ALS genes converge on the endosomal pathway to induce TDP-43 pathology and degeneration.

Frontotemporal dementia and amyotrophic lateral sclerosis (FTD-ALS) are associated with both a repeat expansion in the C9orf72 gene and mutations in the TANK-binding kinase 1 (TBK1) gene. We found that TBK1 is phosphorylated in response to C9orf72 poly(Gly-Ala) [poly(GA)] aggregation and sequestered into inclusions, which leads to a loss of TBK1 activity and contributes to neurodegeneration. When we reduced TBK1 activity using a TBK1-R228H (Arg228→His) mutation in mice, poly(GA)-induced phenotypes were exacerbated. These phenotypes included an increase in TAR DNA binding protein 43 (TDP-43) pathology and the accumulation of defective endosomes in poly(GA)-positive neurons. Inhibiting the endosomal pathway induced TDP-43 aggregation, which highlights the importance of this pathway and TBK1 activity in pathogenesis. This interplay between C9orf72, TBK1, and TDP-43 connects three different facets of FTD-ALS into one coherent pathway.