Petrucelli, Leonard

ABSTRACT Intronic GGGGCC hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Despite its intronic location, this repeat avidly supports synthesis of pathogenic dipeptide repeat (DPR) proteins via repeat-associated non-AUG (RAN) translation. However, the template RNA species that undergoes RAN translation endogenously remains unclear. Using long-read based 5’ RNA ligase-mediated rapid amplification of cDNA ends (5’ Repeat-RLM-RACE), we identified novel C9orf72 transcripts initiating within intron 1 in a C9BAC mouse model, patient-derived iNeurons, and iNeuron-derived polysomes. These cryptic m 7 G-capped mRNAs are at least partially polyadenylated and are more abundant than transcripts derived from intron retention or circular intron lariats. In RAN translation reporter assays, novel intronic template transcripts – even those with short (32 nucleotide) leaders – exhibited robust expression compared to exon-intron and repeat-containing lariat reporters. To assess endogenous lariat repeat RNA contributions to RAN translation, we enhanced endogenous lariat stability by knocking down the lariat debranching enzyme Dbr1. However, this modulation did not impact DPR production in patient-derived iNeurons. These findings identify cryptic, linear, m 7 G-capped intronic-initiating C9orf72 mRNAs as an endogenous template for RAN translation and DPR production, with implications for disease pathogenesis and therapeutic development. SIGNIFICANCE STATEMENT An intronic GGGGCC repeat expansion in C9orf72 supports an unusual translational initiation process known as repeat-associated non-AUG (RAN) translation to produce toxic dipeptide repeat (DPR) proteins that contribute to neurodegeneration in ALS and FTD. How an intronic repeat RNA engages with ribosomes to support such translation is unclear. Here we identify a series of novel mRNA transcripts that initiate within the repeat-containing intron to create linear m 7 G-capped templates for RAN translation from GGGGCC repeats. These cryptic mRNAs are present in patient iNeurons, engage with ribosomes, and robustly support RAN translation. This finding has important implications for both our understanding of the mechanism by which RAN translation occurs and on therapeutic development in this currently untreatable class of neurodegenerative disorders.

ABSTRACT Genetic variation at the TMEM106B locus has been associated with Alzheimer’s disease (AD) and related neurodegenerative phenotypes, yet its haplotypic architecture remains incompletely resolved. We refined the genetic landscape of TMEM106B by integrating AD-GWAS summary statistics (∼978K individuals) with haplotype analyses in 5,873 Dutch genomes including AD-cases, age-matched controls, and cognitively healthy centenarians. In addition to the known linkage disequilibrium (LD) block defining risk and protective haplotypes characterized by the amino acid substitution at residue 185 (Threonine/Serine), we identify a second conditionally independent LD block. Together these form four haplotypes (T1-T4), with T3 strongly enriched in centenarians. Long-read whole-genome sequencing in 493 individuals reveals haplotype-linked structural variation and methylation patterns, including a ∼19 Kbp genomic rearrangement carried by T3. These findings indicate that the TMEM106B locus comprises multiple haplotypes defined by methylation patterns and structural variants, offering insights into GWAS associations with AD risk and preserved cognition at extreme age.

Introduction: Extracranial carotid artery pathology accounts for 15-20% of ischemic strokes. Advancements in magnetic resonance angiography (MRA) with vessel wall imaging (VWI) have enabled the identification of vulnerable plaques, aiding in risk stratification for neurovascular events. This pilot study aimed to identify proteins in plaques with and without vulnerable features on MRA with VWI. Hypothesis: There is a differential proteomic profile of vulnerable and non-vulnerable, thus, these can serve as potential biomarkers for disease progression, stroke prevention, and therapeutic targets. Methods: Consecutive patients undergoing carotid endarterectomy (CEA) were included in the study cohort with pre-operative MRA with VWI. A retrospective chart review was conducted to extract pertinent clinical data including cardiovascular risk factors and medications. The proteomic analysis involved Tandem Mass Tag (TMTpro) labeling of peptides, basic pH HPLC fractionation, and NanoLC-tandem mass spectrometry. Results: A total of sixteen plaques were retrieved from fifteen patients. There were 73.3% male patients. The most common comorbidities were hyperlipidemia (93.3%) and hypertension (80%). 31.2% of patients had symptomatic disease. Eleven plaques were vulnerable in preoperative MRI with VWI. From those, intraplaque hemorrhage was the most common feature (81.1%) . Proteomic analysis revealed 23 relevant proteins significantly elevated in vulnerable plaques, including Proteinase 3 (PRTN3), Phospholipid Transfer Protein (PLTP), and S100 Calcium-Binding Protein A12 (S100A12), with increased abundance exceeding two-fold changes or above (P<0.001). Conversely, three proteins exhibited reduced abundance in vulnerable plaques including Dynamin-3 (DNM3), Transmembrane Protein 181 (TMEM181), and Adducin-3 (ADD3) (P<0.05). All results are summarized in Figure 1A-C and Table I and II. Conclusion: This study contributes to the understanding of protein biomarkers associated with carotid plaque vulnerability, offering insights into disease progression, stroke prevention, and identification of potential therapeutic targets (Figure 1D) . Furthermore, these patients could benefit from early surgical intervention if these biomarkers were to be detected. We present a comprehensive study of multiple proteins with significant bioavailability in explanted plaque specimens that can serve as a foundation for identifying blood biomarkers for future validation studies.

AbstractINTRODUCTIONPlasma amyloid beta42/amyloid beta40 (Aβ42/Aβ40) and phosphorylated tau217 (p‐tau217) identify individuals with primary Alzheimer's disease (AD). They may detect AD co‐pathology in the setting of other primary neurodegenerative diseases, but this has not been systematically studied.METHODSWe compared the clinical, neuroimaging, and neuropathological associations of plasma Aβ42/Aβ40 (mass spectrometry), p‐tau217 (electrochemiluminescence), and neurofilament light ([NfL], single molecule array [Simoa]), as markers of AD co‐pathology, in a sporadic frontotemporal dementia (FTD) cohort (n = 620).RESULTSAβ42/Aβ40 showed no clinicopathological associations. High p‐tau217 was present in amnestic dementia (AmD) presumed to be due to FTD, logopenic primary progressive aphasia (lvPPA), and APOEε4 carriers, and correlated with worse baseline and longitudinal clinical scores, lower hippocampal volumes, and more severe AD co‐pathology (Braak Stage). NfL was elevated in all FTD phenotypes, and correlated with clinical scores and frontotemporal brain volumes.DISCUSSIONPlasma p‐tau217 has clinical, neuroimaging, and neuropathological correlates in sporadic FTD and may identify FTD cases with AD co‐pathology.Highlights Alzheimer's disease (AD) features could be identified with plasma phosphorylated tau217 (p‐tau217) in frontotemporal lobar degeneration (FTLD). Plasma p‐tau217 is a better discriminator of AD co‐pathology and AD‐associated features in FTLD than plasma amyloid beta42/amyloid beta40 (Aβ42/Aβ40) and neurofilament light (NfL). In FTLD, plasma p‐tau217, but not Aβ42/Aβ40 or neurofilament light, has phenotypical, neurocognitive, and neuroimaging correlates suggestive of AD co‐pathology.

Abstract Background Perry syndrome (PS) is a rare and fatal hereditary autosomal dominant neurodegenerative disorder caused by mutations in dynactin ( DCTN1 ). PS brains accumulate inclusions positive for ubiquitin, transactive‐response DNA‐binding protein of 43 kDa (TDP‐43), and to a lesser extent dynactin. Objectives Little is known regarding the contributions of TDP‐43, an RNA binding protein that represses cryptic exon inclusion, in PS. Therefore, we sought to identify the degree of TDP‐43 dysfunction in two regions of PS brains. Methods We evaluated the levels of insoluble pTDP‐43 and TDP‐43‐regulated cryptic RNAs and protein in the caudate nucleus and substantia nigra of 7 PS cases, 12 cases of frontotemporal lobar degeneration (FTLD) with TDP‐43 pathology, and 11 cognitively healthy controls without TDP‐43 pathology. Results Insoluble pTDP‐43 protein levels were detected in PS brains to a similar extent in the caudate nucleus and substantia nigra but lower than those in FTLD brains. The caudate nucleus of PS showed accumulation of eight TDP‐43‐regulated cryptic RNAs ( ACTL6B , CAMK2B , STMN2 , UNC13A , KCNQ2 , ATG4B , GPSM2 , and HDGFL2 ) and cryptic protein (HDGFL2) characteristic of FTLD. Conversely, only one cryptic target, UNC13A , reached significance in the substantia nigra despite similar pTDP‐43 levels. Conclusion We detected TDP‐43 cryptic RNAs and protein in PS caudate nucleus. Given the importance of cryptic exon biology in the development of biomarkers, and the identification of novel targets for therapeutic intervention, it is imperative we understand the consequences of TDP‐43 dysfunction across different brain regions and determine the targets that are specific and common to TDP‐43 proteinopathies. © 2025 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

AbstractBackgroundAs new anti‐amyloid immunotherapies emerge for Alzheimer’s disease (AD), it is clear that early diagnosis of AD pathology is crucial for treatment success. This can be challenging in atypical presentations of AD and, together with our reliance on CSF or PET scans, can, at times, lead to delayed diagnosis. Here, we further explore the possible role of plasma tau phosphorylated at threonine 217 (P‐tau217) for the detection of primary AD or AD co‐pathology when frontotemporal dementia spectrum disorders are the main clinical presentation.MethodParticipants were recruited through ALLFTD, a North American multisite research consortium for the longitudinal assessment of frontotemporal lobar degeneration. After excluding cases with known FTD‐causing mutations, 573 participants with sporadic disease, mild cognitive/behavioral impairment, or healthy familial controls evaluated between 2014 and 2023 were included in this analysis (45.8% female, median age 66 years). 39 of those cases underwent neuropathological evaluation. Plasma P‐tau217 was measured with the Eli Lilly electrochemiluminescence‐based assay. P‐tau217 concentrations were compared by phenotype, disease severity, genotype, and, when available, neuropathological diagnosis with non‐parametric tests. Its diagnostic performance was tested with ROC curves. Associations between plasma P‐tau217 concentrations and clinical scales of global cognition, memory, executive function, motor function, and social cognition with linear regressions and non‐parametric tests.ResultPlasma P‐tau217 concentrations were higher in logopenic variant primary progressive aphasia (lvPPA) and amnestic dementia (AmDem), compared to participants with normal cognition or other FTD phenotypes. P‐tau217 concentrations were higher in APOE e4 carriers, compared to non‐carriers, regardless of phenotype, but were not affected by disease severity. P‐tau217 concentrations showed associations with clinical measures of global cognition, memory, and executive function, but had no associations with measures of motor function or social cognition. Plasma P‐tau217 concentrations were elevated in high and intermediate ADNC scoring, or more advanced Braak stages, even when AD was not the primary pathology.ConclusionEven when FTD is suspected, high plasma p‐tau 217 concentrations are strongly associated with underlying AD as primary pathology or contributing co‐pathology. Plasma P‐tau217 could be a tool to support the development of disease‐modifying therapies for atypical Alzheimer’s disease presentations or for FTD cases with Alzheimer’s disease co‐pathology.

AbstractBackgroundGrowth Differentiation Factor 15 (GDF15) is a TGF‐beta superfamily protein upregulated during immune‐mediated stress responses. Peripheral GDF15 is linked to adverse aging across organ systems including the brain. Immune dysregulation is implicated in frontotemporal dementia (FTD) pathogenesis. We examined how plasma GDF15 levels associated with longitudinal cognitive and neurofilament light chain (NfL) trajectories in adults with FTD and controls. Furthermore, we tested the utility of GDF15 as a prognostic biomarker against common inflammatory proteins (CRP and IL6).Method231 adults with FTD (24.35% sporadic FTD syndromes; 19.19% MAPT; 19.56% GRN; 22.14% C9orf72 pathogenic carriers) and 40 normal controls from the ALLFTD Consortium completed longitudinal neuropsychological testing and a blood draw with plasma analyzed on SomaScan V4.0 (baseline only: GDF15, CRP, and IL6) and Simoa (longitudinal: NfL). CRP and IL6 were selected as control markers to test the specificity of GDF15. Linear mixed effects models separately examined longitudinal cognition (global, executive functioning, memory, and language z‐scores) and plasma NfL levels as a function of baseline plasma GDF15, time, and their interaction, controlling for baseline age, sex, and education. Parallel models were conducted for CRP and IL6.ResultIn the whole cohort, higher baseline plasma GDF15 levels associated with steeper global cognitive decline (b = ‐3.91, p = 0.0092) and NfL accumulation (b = 3.88, p = 0.00088) over time. When examining cognitive domains, we found GDF15 only associated with worse executive functioning trajectories (b = ‐3.19, p = 0.0021). In contrast, baseline levels of CRP and IL6 did not display statistically significant relationships with cognitive or NfL trajectories. Prognostic effects of GDF15 remained statistically significant when restricting analyses to those with FTD pathogenic variants (b = ‐2.98, p = 0.0054 [global cognition], b = 2.69, p = 0.0098 [NfL]).ConclusionAdults with FTD and higher baseline peripheral GDF15 levels exhibit accelerated cognitive decline and neurodegeneration. Our results support GDF15 as a potential prognosticator of adverse clinical outcomes in FTD and highlight the relevance of GDF15‐specific pathways in FTD progression. Plasma GDF15 may provide utility over commonly examined inflammatory proteins and further work to explore its clinical value is indicated.

AbstractBackgroundIdentification of useful fluid biomarkers is expected to advance frontotemporal dementia spectrum disorders (FTD) care and therapeutic development. The clinical utility of emerging plasma Amyloid, Tau, and Neurodegeneration (ATN) biomarkers in FTD remains unexplored. This study analyzed ATN patterns by sporadic FTD phenotype, based on amyloid beta1‐42 (Aβ1‐42), amyloid beta1‐40 (Aβ1‐40), phospho‐tau217 (p‐tau217), and neurofilament (NfL) plasma concentrations.MethodThe present study included 625 participants enrolled in ALLFTD (46% female, median age 61 ± 12 years) for whom biomarker data were available. Plasma Aβ1‐42/Aβ1‐40 measures were determined by immunoprecipitation‐mass spectrometry whereas plasma p‐tau217 and NfL concentrations were respectively determined by electrochemiluminescence‐ and fluorescence‐based immunoassays. Biomarker concentrations were compared by phenotype and disease severity with ANOVA. Their diagnostic performance was tested with Receiver Operating Characteristic (ROC) curves, and biomarker relationships with measures of clinical severity were tested through linear regressions controlling for age, sex, and APOE genotype.ResultHigher plasma p‐tau217 and p‐tau217/Aβ1‐42, levels were observed in logopenic primary progressive aphasia (lvPPA) and amnestic dementia (AmD) cohorts, compared to other FTD groups. These biomarkers were not associated with disease severity but were affected by APOE4 genotype. High plasma NfL level was observed in fully symptomatic patients compared to controls, and as a function of disease severity. Plasma Aβ1‐42/Aβ1‐40 level was not different between phenotypes and was not affected by clinical severity. P‐tau217 level, but not Aβ1‐42/Aβ1‐40 or NfL levels showed high discrimination of lvPPA and AmD from other phenotypes (AUC = 0.87 (95% CI 0.77 – 0.96, p < 0.0001, 73% sensitivity, 92% specificity). The p‐tau217/Aβ1‐42 ratio improved this discrimination (AUC = 0.9, 95% CI 0.83 – 1, p < 0.0001, 77% sensitivity, 94% specificity). Plasma Aβ1‐42/Aβ1‐40, p‐tau217, NfL, p‐tau217/Aβ1‐42, p‐tau217/NfL, and (Aβ1‐42 x p‐tau217)/NfL were associated with cognitive (MoCA, CVLT), motor (UPDRS), and social behavior (RSMS) performances at baseline.ConclusionPlasma p‐tau217/Aβ1‐42 identifies, with high specificity, two phenotypes associated with Alzheimer’s disease: lvPPA and AmD. Pending molecular neuroimaging and neuropathological confirmation, this biomarker could be of value to identify primary Alzheimer’s disease pathology or co‐pathology associated with Alzheimer’s disease when sporadic FTD is suspected.

AbstractBackgroundTransactive response DNA‐binding protein of 43 kDa (TDP‐43) is implicated in numerous neurodegenerative diseases, including Alzheimer’s disease (AD). Immunohistochemically stained TDP‐43 is linked to faster rates of hippocampal atrophy (HA) in AD. However, it is unclear whether phosphorylated TDP‐43 (pTDP‐43) measured using an immunoassay is also associated with HA. We explored associations between pTDP‐43 levels, TDP‐43 immunohistochemistry, and annual rate of HA in AD. We hypothesized that higher pTDP‐43 levels would be associated with positive TDP‐43 immunohistochemistry, and faster rates of HA.MethodThirty‐seven cognitively impaired participants from the Mayo Clinic Alzheimer’s Disease Research Center underwent serial brain MRIs and brain autopsy. Hippocampal TDP‐43 was detected histochemically, and pTDP‐43 was quantified using biochemical assay. Hippocampal volumes were measured using the longitudinal pipeline in FreeSurfer‐7 and annualized HA rates were calculated. Linear regression analyzed the relationship between HA and pTDP‐43 biochemical assay levels. pTDP‐43 was categorized as positive (≥400) or negative (<400), and logistic regression tested the association of these levels with HA rates. The correlation between TDP‐43 immunohistochemistry status and biochemical assay levels was evaluated using descriptive statistics. All analyses were adjusted for potential confounders.ResultsThe mean age at death was 83.5 (±9.9) years with 49% females. Median scan interval was 3‐years. A negative relationship was observed between age at death and annual HA rate (β=‐3.1; 95% CI [‐5.3,‐0.9]; p=0.007), and a positive relationship was observed between pTDP‐43 level and annual HA rate (β=0.02; 95% CI [0.003,0.03]; p=0.015). A one‐unit increase in HA rate was associated with a 3% increase in the odds of being pTDP‐43 positive (≥400) (odds ratio=1.03; CI [1.007,1.05]; p=0.014). Cases that tested positive for Immunohistochemistry TDP‐43 had significantly higher mean pTDP‐43 immunoassay levels (1508 ± 2072 vs. 202 ± 131; p=0.008). More cases with positive IHC TDP‐43 surpassed the pTDP‐43 immunoassay cut‐off level of 400 vs the negative participants (p=0.003).ConclusionHigher pTDP‐43 levels in the hippocampus are linked to faster HA in AD and correlated with immunohistochemistry TDP‐43 positivity. These findings underscore the role of TDP‐43 in AD neurodegeneration. The biological and clinical implications of these observations necessitate further exploration in subsequent studies.