Autophagosome-targeting single-domain antibody clears tau in patient-derived neurons and improves motor function in tauopathy mice.

Tauopathies are neurodegenerative diseases characterized by pathological tau accumulation, leading to motor and neuropsychiatric symptoms. Effective tau-targeting therapies remain a major challenge. Here, we present 1D9-LIRΔTP53INP2, a single-domain antibody (sdAb)-based protein degrader that facilitates tau clearance via the autophagy-lysosomal pathway. This engineered molecule combines the anti-tau sdAb 1D9 with an LC3-interacting region (LIRΔTP53INP2) to promote autophagosomal recruitment, mimicking autophagy receptors by simultaneously binding tau and LC3. In frontotemporal dementia (FTD) patient-derived neurons and JNPL3 tauopathy mice, both harboring the P301L tau mutation, 1D9-LIRΔTP53INP2 significantly reduced tau levels and improved motor function in mice. These findings underscore the therapeutic potential of sdAb-based protein degraders for tauopathies. Given the challenges of brain delivery for conventional antibodies, sdAbs with enhanced brain penetration and efficacy offer a promising strategy for treatment of neurodegenerative diseases.

New unexpected role for Wolfram Syndrome protein WFS1: a novel therapeutic target for Alzheimer's disease?

Tauopathies are neurodegenerative diseases characterized by aberrant forms of tau protein accumulation leading to neuronal death in focal brain areas. Positron emission tomography (PET) tracers that bind to pathological tau are used in diagnosis, but there are no current therapies to eliminate these tau species. We employed targeted protein degradation technology to convert a tau PET-probe into a functional degrader of pathogenic tau. The hetero-bifunctional molecule QC-01–175 was designed to engage both tau and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, to trigger tau ubiquitination and proteasomal degradation. QC-01–175 effected clearance of tau in frontotemporal dementia (FTD) patient-derived neuronal cell models, with minimal effect on tau from neurons of healthy controls, indicating specificity for disease-relevant forms. QC-01–175 also rescued stress vulnerability in FTD neurons, phenocopying CRISPR-mediated MAPT-knockout. This work demonstrates that aberrant tau in FTD patient-derived neurons is amenable to targeted degradation, representing an important advance for therapeutics.

Tauopathies are neurodegenerative diseases characterized by aberrant forms of tau protein accumulation leading to neuronal death in focal brain areas. Positron emission tomography (PET) tracers that bind to pathological tau are used in diagnosis, but there are no current therapies to eliminate these tau species. We employed targeted protein degradation technology to convert a tau PET-probe into a functional degrader of pathogenic tau. The hetero-bifunctional molecule QC-01-175 was designed to engage both tau and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, to trigger tau ubiquitination and proteasomal degradation. QC-01-175 effected clearance of tau in frontotemporal dementia (FTD) patient-derived neuronal cell models, with minimal effect on tau from neurons of healthy controls, indicating specificity for disease-relevant forms. QC-01-175 also rescued stress vulnerability in FTD neurons, phenocopying CRISPR-mediated MAPT-knockout. This work demonstrates that aberrant tau in FTD patient-derived neurons is amenable to targeted degradation, representing an important advance for therapeutics.

BackgroundTargeted protein degradation is an attractive modality of therapeutic intervention for a wide range of neurodegenerative diseases. Intervention at the protein level is highly desired since pathogenic changes such as conformational change or post‐translational modifications can be directly targeted.MethodWe designed protein constructs that contain an intracellular binding domain for targeting tau fused to a domain for recruiting the ubiquitination machinery. We expressed these constructs in a cultured human cell line and dissociated cultures of mouse hippocampal neurons expressing human tau. We assessed the degradation of human tau using fluorescence microscopy and by tau quantification using immunoblotting.ResultWe found that the ubiquitin ligase substrate adaptor speckle‐type POZ protein (SPOP), when fused to camelid‐derived single‐domain antibody fragments (nanobodies), allows efficient degradation of nanobody binding proteins imported to the nucleus. We found that the endogenous SPOP domain contains a strong nuclear localization signal, resulting in nuclear accumulation and aggregation of tau. Based on this we hypothesized that the strong nuclear localization signal overloaded the intracellular protein degradation capacity, disrupting the cellular homeostasis. To test this, we engineered SPOP variants with altered nuclear transport efficiency. The engineered variants altered the degree of tau degradation and nuclear accumulation. Interestingly, this regulated degradation approach resulted in preferential degradation of tau dissociated from the microtubule, leading to nearly exclusive colocalization of tau to the microtubule in mammalian cells and primary mouse hippocampal neurons.ConclusionThe new constructs developed here allow fine tuning of nuclear translocation rate and degradation efficiency of tau. This controlled degradation strategy may be beneficial in developing therapeutic approaches for regulated tau degradation without overloading the proteasomal capacity.

Autophagic degradation of tau in primary neurons and its enhancement by trehalose

Neuropsychiatric symptoms (NPS) are prevalent in neurodegenerative disorders, however, their frequency and impact on function across different disorders is not well understood. We compared the frequency and severity of NPS across Alzheimer's disease (AD) (either with mild cognitive impairment or dementia), Cerebrovascular disease (CVD), Parkinson's disease (PD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS), and explored the association between NPS burden and function. We obtained data from Ontario Neurodegenerative Disease Research Initiative (ONDRI) that included following cohorts: AD (N = 111), CVD (N = 148), PD (N = 136), FTD (N = 50) and ALS (N = 36). We compared the frequency and severity of individual NPS (assessed by the neuropsychiatric inventory questionnaire) across cohorts using generalized estimating equations and analysis of variance. Second, we assessed the relationship of NPS burden with instrumental (iADLs) and basic (ADLs) activities of living across cohorts using multivariate linear regression while adjusting for relevant demographic and clinical covariates. Frequency of NPS varied across cohorts (χ2(4) = 34.4, p < .001), with post-hoc tests showing that FTD had the greatest frequency as compared to all other cohorts. The FTD cohort also had the greatest severity of NPS (H(4) = 34.5, p < .001). Further, there were differences among cohorts in terms of the association between NPS burden and ADLs (F(4,461) = 3.1, p = 0.02). Post-hoc comparisons suggested that this finding was driven by the FTD group, however, the differences did not remain significant following Bonferroni correction. There were no differences among cohorts in terms of the association between NPS burden and IADLs. NPS frequency and severity are markedly greater in FTD as compared to other neurodegenerative diseases. Further, NPS burden appears to be associated differently with function across neurodegenerative disorders, highlighting the need for individualized clinical interventions.

The microtubule-associated protein tau, which becomes hyperphosphorylated and pathologically aggregates in a number of these diseases, is extremely sensitive to manipulations of chaperone signaling. For example, Hsp90 inhibitors can reduce the levels of tau in transgenic mouse models of tauopathy. Because of this, we hypothesized that a number of Hsp90 accessory proteins, termed co-chaperones, could also affect tau stability. Perhaps by identifying these co-chaperones, new therapeutics could be designed to specifically target these proteins and facilitate tau clearance. Here, we report that the co-chaperone Cdc37 can regulate aspects of tau pathogenesis. We found that suppression of Cdc37 destabilized tau, leading to its clearance, whereas Cdc37 overexpression preserved tau. Cdc37 was found to co-localize with tau in neuronal cells and to physically interact with tau from human brain. Moreover, Cdc37 levels significantly increased with age. Cdc37 knockdown altered the phosphorylation profile of tau, an effect that was due in part to reduced tau kinase stability, specifically Cdk5 and Akt. Conversely, GSK3β and Mark2 were unaffected by Cdc37 modulation. Cdc37 overexpression prevented whereas Cdc37 suppression potentiated tau clearance following Hsp90 inhibition. Thus, Cdc37 can regulate tau in two ways: by directly stabilizing it via Hsp90 and by regulating the stability of distinct tau kinases. We propose that changes in the neuronal levels or activity of Cdc37 could dramatically alter the kinome, leading to profound changes in the tau phosphorylation signature, altering its proteotoxicity and stability.

BackgroundSynucleinopathies and tauopathies are characterized by progressive deposition of α‐synuclein (α‐syn) and tau proteins, respectively. These diseases have been linked to proteins mutations or excess α‐syn gene, indicating prominent roles of these proteins in their etiology and pathogenesis. Consequently, being able to non‐invasively image these aggregates would allow early and accurate diagnosis to facilitate clinical trials on drugs targeting these lesions and eventually for prophylactic therapies. Recently, several dye‐based imaging probes with selectivity for tau aggregates have been developed but suitable imaging biomarkers for synucleinopathies are still unavailable. However, these β‐sheet binders bind to different amyloids and thereby lack specificity. Compared to β‐sheet dyes, single domain antibodies (sdAbs), found in camelids and a few other species, are highly specific and their small size allows better brain entry and distribution than whole antibodies.MethodTo develop anti‐α‐syn or anti‐tau sdAbs, we immunized llamas with α‐syn and tau preparations, respectively. Phage display libraries were generated from peripheral blood mononuclear cells and screened for binding to α‐syn or tau proteins using various assays, resulting in prototype sdAbs deemed promising for in vivo imaging. Subsequently, these anti‐α‐syn or anti‐tau sdAbs were labeled with a near‐infrared tag and injected intravenously in synucleinopathy, tauopathy or control mice, followed by imaging in an In Vivo Imaging System (IVIS). Subsequently, brains were extracted for tissue analysis.ResultIntravenous injection of labeled anti‐α‐syn or anti‐tau sdAbs resulted in strong in vivo brain signal detected through the intact head in synucleinopathy and tauopathy mice, respectively, but not in control mice. Notably, anti‐α‐syn sdAbs did not give signal in tauopathy mice and vice versa. The in vivo brain signal correlated strongly with insoluble, soluble, and phospho‐antigen within the brain. Postmortem analysis revealed extensive co‐localization of the sdAb imaging probe with α‐syn or tau aggregates within neurons in the endosomal‐lysosomal system, indicating their interaction in these degradation pathways.ConclusionThese anti‐α‐syn or anti‐tau sdAb probes allow non‐invasive and specific in vivo imaging of α‐synuclein vs tau pathology in mice, with brain signals correlating strongly with lesion burden. These small antibody derivatives have great potential for in vivo diagnosis of these diseases.

Handedness has been shown to be associated with genetic variation involving brain development and neuropsychiatric diseases. Whether handedness plays a role in clinical phenotypes of common neurodegenerative diseases has not been extensively studied. This study used the National Alzheimer’s Coordinating Center database to examine whether self-reported handedness was associated with neuropsychological performance and neuropsychiatric symptoms in cognitively unimpaired individuals (n = 17 670), individuals with Alzheimer’s disease (n = 10 709), behavioural variant frontotemporal dementia (n = 1132) or dementia with Lewy bodies (n = 637). Of the sample, 8% were left-handed, and 2% were ambidextrous. There were small differences in the handedness distributions across the cognitively unimpaired, Alzheimer’s disease, behavioural variant frontotemporal dementia and dementia with Lewy bodies groups (7.2–9.5% left-handed and 0.9–2.2% ambidextrous). After adjusting for age, gender and education, we found faster performance in Trail Making Test A in cognitively unimpaired non-right-handers (ambidextrous and left-handed) compared with right-handers. Excluding ambidextrous individuals, the left-handed cognitively unimpaired individuals had faster Trail Making Test A performance and better Number Span Forward performance than right-handers. Overall, handedness had no effects on most neuropsychological tests and none on neuropsychiatric symptoms. Handedness effect on Trail Making Test A in the cognitively unimpaired is likely to stem from test artefacts rather than a robust difference in cognitive performance. In conclusion, handedness does not appear to affect neuropsychological performance or neuropsychiatric symptoms in common neurodegenerative diseases.

Complex genetic relationships between neurodegenerative disorders and neuropsychiatric symptoms have been shown, suggesting shared pathogenic mechanisms and emphasizing the potential for developing common therapeutic targets. Apolipoprotein E (APOE) genotypes and their corresponding protein (ApoE) isoforms may influence the biophysical properties of the cell membrane lipid bilayer. However, the role of APOE in central nervous system pathophysiology extended beyond its lipid transport function. In the present review article, we analyzed the links existing between APOE genotypes and the neurobiology of neuropsychiatric symptoms in neurodegenerative and vascular diseases. APOE genotypes (APOE ε2, APOE ε3, and APOE ε4) were implicated in common mechanisms underlying a wide spectrum of neurodegenerative diseases, including sporadic Alzheimer’s disease, synucleinopathies such as Parkinson’s disease and Lewy body disease, stroke, and traumatic brain injury. These shared pathways often involved neuroinflammation, abnormal protein accumulation, or responses to acute detrimental events. Across these conditions, APOE variants are believed to contribute to the modulation of inflammatory responses, the regulation of amyloid and tau pathology, as well as the clearance of proteins such as α-synuclein. The bidirectional interactions among ApoE, amyloid and mitochondrial metabolism, immunomodulatory effects, neuronal repair, and remodeling underscored the complexity of ApoE’s role in neuropsychiatric symptoms associated with these conditions since from early phases of cognitive impairment such as mild cognitive impairment and mild behavioral impairment. Besides ApoE-specific isoforms’ link to increased neuropsychiatric symptoms in Alzheimer’s disease (depression, psychosis, aberrant motor behaviors, and anxiety, not apathy), the APOE ε4 genotype was also considered a significant genetic risk factor for Lewy body disease and its worse cognitive outcomes. Conversely, the APOE ε2 variant has been observed not to exert a protective effect equally in all neurodegenerative diseases. Specifically, in Lewy body disease, this variant may delay disease onset, paralleling its protective role in Alzheimer’s disease, although its role in frontotemporal dementia is uncertain. The APOE ε4 genotype has been associated with adverse cognitive outcomes across other various neurodegenerative conditions. In Parkinson’s disease, the APOE ε4 allele significantly impacted cognitive performance, increasing the risk of developing dementia, even in cases of pure synucleinopathies with minimal co-pathology from Alzheimer’s disease. Similarly, in traumatic brain injury, recovery rates varied, with APOE ε4 carriers demonstrating a greater risk of poor long-term cognitive outcomes and elevated levels of neuropsychiatric symptoms. Furthermore, APOE ε4 influenced the age of onset and severity of stroke, as well as the likelihood of developing stroke-associated dementia, potentially due to its role in compromising endothelial integrity and promoting blood–brain barrier dysfunction.

BackgroundFalls are the most common injury faced by older adults and those with neurodegenerative diseases. Falls can result in concussion/mild traumatic brain injury(mTBI). Concussions in older adults or those with neurodegenerative disease can have a significant impact on behavior as post‐concussion symptoms include neuropsychiatric issues. We hypothesized that there is a relationship between past fall and neuropsychiatric symptoms and neuropsychiatric symptom severity.MethodsWe used data on falls and Neuropsychiatric Inventory (NPI) from the Ontario Neurodegenerative Disease Research Initiative dataset for 480 individuals with neurodegenerative diseases (Alzheimer’s Disease, Parkinson’s Disease, Amyotrophic lateral sclerosis, frontotemporal dementia and vascular cognitive impairment). We used the Chi‐squared and Mann‐Whitney tests to compare frequency of NPI symptoms (anxiety, depression, irritability, disinhibition, apathy, delusions, hallucinations, agitation, euphoria, motor‐disturbance, night‐time behaviour, appetite), and total NPI severity and distress, respectively, between patients with and without falls in the past 12 months.ResultsComparing patients with falls (n = 169; mean‐age = 68.3±9; 36% F) to patients without falls (n = 311; mean‐age = 68.7±7; 32% F), there was a significantly higher frequency of anxiety (Chi‐squared test, X2 (df = 1, N = 480) = 12.859, P‐value = 0.0003); higher median anxiety severity (Mann‐Whitney/Wilcoxon‐test p‐value = 0.0002); and higher median partner anxiety distress (Wilcox test p‐value = 0.0006) in those who had had a previous fall compared to those who had not, even with multiple comparison correction. Depression, apathy, disinhibition, night‐time behaviours, and eating/appetite changes and total NPI severity were significantly worse in those with previous falls but did not survive multiple comparison correction.ConclusionWe found that anxiety frequency, severity and distress were much higher in patients with neurodegenerative disease who had a fall in the preceding 12 months compared to those without falls. Our study suggests that neuropsychiatric symptoms, especially anxiety are frequent and should be assessed in those with previous falls as they can be a consequence of mild brain injury and may contribute to worsening cognition or behaviors.

Cavum septum pellucidum (CSP) is commonly observed upon neuroimaging examination in individuals exposed to repetitive head impacts (RHI) and post-mortem in cases with chronic traumatic encephalopathy. Consequently, CSP has been proposed as a potential biomarker for RHI-related neurodegeneration, yet prevalence estimates of CSP across other neurodegenerative diseases and its clinical implications are largely unknown. We assessed CSP prevalence and clinical correlates in individuals with RHI exposure, a history of traumatic brain injury (TBI), a neurodegenerative disease (i.e. Alzheimer's disease or frontotemporal dementia) and normal cognition. The primary group of interest, i.e. individuals exposed to RHI in contact sports or military service (n = 65; mean exposure 21.58 years), was compared against age- and sex-matched participants with TBI (n = 57; number of TBI range: 1-5) and non-exposed participants of the Amsterdam Dementia Cohort (Alzheimer's disease, n = 30; frontotemporal dementia, n = 24; normal cognition, n = 27). Structural 3D brain MRI scans were visually rated for CSP grade (ranging 0-4) by two raters blinded to the clinical information. A CSP of at least Grade 2 was considered enlarged/abnormal. Inter-rater reliability was assessed with Cohens' weighted Kappa (κ). We investigated whether prevalence of enlarged CSP differed between groups and assessed associations with neuropsychological outcomes (verbal memory, processing speed, mental flexibility and semantic fluency), neuropsychiatric symptoms (neuropsychiatric inventory), ventricular enlargement as measured with Evan's index and MRI volumes of composite regions (limbic, temporal-meta regions and the whole brain). Inter-rater reliability was substantial [κ = 0.734 (95% confidence interval 0.67-0.80)]. An enlarged CSP was more often observed in the RHI group (44.6%) compared with individuals with Alzheimer's disease [13.3%, odds ratio (OR) = 5.24 (1.79-19.26)], frontotemporal dementia [16.7%, OR = 4.03 (1.35-15.02)] and normal cognition [18.5%, OR = 3.54 (1.27-11.62)], all P FDR < 0.05, but not compared with the TBI group [29.8%, OR = 1.90 (0.90-4.06), P FDR = 0.094]. In those with RHI, enlarged CSP was associated with lower outcomes on verbal memory learning (η² = 0.09, P FDR = 0.023) and recall (η² = 0.08, P FDR = 0.030). For TBI, enlarged CSP was associated with lower performance on verbal memory learning; however, this lost significance after multiple comparison correction (η² = 0.014, P FDR = 0.09). Enlarged CSP was not associated with the composite MRI volumes, ventricular enlargement or neuropsychiatric symptoms. In summary, enlarged CSP was more prevalent in RHI-exposed individuals compared with individuals with a neurodegenerative disease or normal cognition, but not compared with TBI, and was associated with lower verbal memory performance in the RHI group. Our study highlights enlarged CSP as a potential consequence of long-term head impact exposure and, to a lesser extent, TBI, rather than a general consequence of neurodegeneration.

We report on a novel transgenic mouse model expressing human full-length Tau with the Tau mutation A152T (hTau(AT)), a risk factor for FTD-spectrum disorders including PSP and CBD Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis-sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short- or long-term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage-gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau(AT) causes excitotoxicity mediated by NR2B-containing NMDA receptors due to enhanced extracellular glutamate.

Tauopathies are a spectrum of diseases characterized by the pathological aggregation of tau proteins. Several therapeutic strategies have been developed to treat or stop the progression of these diseases, but all have failed in clinical trials. One potential reason for these failures is that disease-causing proteins are resilient to treatment with conventional drugs since they lack a predefined monomeric structure and an active binding site. Tau is an intrinsically disordered protein; paradoxically, its flexible conformation makes it an ideal candidate for targeted protein degradation (TPD) approaches, which bypass the need for structured binding pockets by inducing proximity-based recruitment to degradative machinery. TPD uses bifunctional molecules to recruit proteins to the ubiquitin-proteasome system (UPS) or autophagy-lysosomal pathways, overcoming limitations of traditional small-molecule inhibitors (SMIs). As these technologies have been effective in degrading several disease-related proteins, they hold significant promise for treating tauopathies caused by protein aggregation. Herein, we review the tau structure and functions, summarize the main post-translational modifications (PTMs) of tau including those causing pathological aggregation of tau, the major degradative cellular machinery and their defects in pathological state, and discuss the advantages and current progress of targeted protein degradation strategies compared to traditional approaches.