Single-domain antibody-based protein degrader for synucleinopathies.

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

Neuronal hypofunction and network dysfunction in a mouse model at an early stage of tauopathy.

We previously reported altered neuronal Ca 2+ dynamics in the motor cortex of 12-month-old JNPL3 tauopathy mice during quiet wakefulness or forced running, with a tau antibody treatment significantly restoring the neuronal Ca 2+ activity profile and decreasing pathological tau in these mice 1 . Whether neuronal functional deficits occur at an early stage of tauopathy and if tau antibody treatment is effective in younger tauopathy mice needed further investigation. In addition, neuronal network activity and neuronal firing patterns have not been well studied in behaving tauopathy models. In this study, we first performed in vivo two-photon Ca 2+ imaging in JNPL3 mice in their early stage of tauopathy at 6 months of age, compared to 12 month old mice and age-matched wild-type controls to evaluate neuronal functional deficits. At the animal level, frequency of neuronal Ca 2+ transients decreased only in 6 month old tauopathy mice compared to controls, and only when animals were running on a treadmill. The amplitude of neuronal transients decreased in tauopathy mice compared to controls under resting and running conditions in both age groups. Total neuronal activity decreased only in 6 month old tauopathy mice compared to controls under resting and running conditions. Within either tauopathy or wild-type group, only total activity decreased in older wild-type animals. The tauopathy mice at different ages did not differ in neuronal Ca 2+ transient frequency, amplitude or total activity. In summary, neuronal function did significantly attenuate at an early age in tauopathy mice compared to controls but interestingly did not deteriorate between 6 and 12 months of age. A more detailed populational analysis of the pattern of Ca 2+ activity at the neuronal level in the 6 month old cohort confirmed neuronal hypoactivity in layer 2/3 of primary motor cortex, compared to wild-type controls, when animals were either resting or running on a treadmill. Despite reduced activity, neuronal Ca 2+ profiles exhibited enhanced synchrony and dysregulated responses to running stimulus. Further ex vivo electrophysiological recordings revealed reduction of spontaneous excitatory synaptic transmission onto and in pyramidal neurons and enhanced excitability of inhibitory neurons in motor cortex, which were likely responsible for altered neuronal network activity in this region. Lastly, tau antibody treatment reduced pathological tau and gliosis partially restored the neuronal Ca 2+ activity deficits but failed to rescue altered network changes. Taken together, substantial neuronal and network dysfunction occurred in the early stage of tauopathy that was partially alleviated with acute tau antibody treatment, which highlights the importance of functional assessment when evaluating the therapeutic potential of tau antibodies. Highlights: Layer 2/3 motor cortical neurons exhibited hypofunction in awake and behaving mice at the early stage of tauopathy.Altered neuronal network activity disrupted local circuitry engagement in tauopathy mice during treadmill running.Layer 2/3 motor cortical neurons in tauopathy mice exhibited enhanced neuronal excitability and altered excitatory synaptic transmissions.Acute tau antibody treatment reduced pathological tau and gliosis, and partially restored neuronal hypofunction profiles but not network dysfunction.

Single-Domain Antibody-Based Protein Degrader for Synucleinopathies.

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

Tau-targeting therapies for Alzheimer disease: current status and future directions.

Alzheimer disease (AD) is the most common cause of dementia in older individuals. AD is characterized pathologically by amyloid-ß (Aß) plaques and tau neurofibrillary tangles in the brain, with associated loss of synapses and neurons, which eventually results in dementia. Many of the early attempts to develop treatments for AD focused on Aß, but a lack of efficacy of these treatments in terms of slowing disease progression led to a change of strategy towards targeting of tau pathology. Given that tau shows a stronger correlation with symptom severity than does Aß, targeting of tau is more likely to be efficacious once cognitive decline begins. Anti-tau therapies initially focused on post-translational modifications, inhibition of tau aggregation and stabilization of microtubules. However, trials of many potential drugs were discontinued because of toxicity and/or lack of efficacy. Currently, the majority of tau-targeting agents in clinical trials are immunotherapies. In this Review, we provide an update on the results from the initial immunotherapy trials and an overview of new therapeutic candidates that are in clinical development, as well as considering future directions for tau-targeting therapies.

An Autopsy Series of Seven Cases of VPS13A Disease (Chorea-Acanthocytosis).

BACKGROUND: Vacuolar protein sorting 13 homolog A (VPS13A) disease, historically known as chorea-acanthocytosis, is a rare neurodegenerative disorder caused by biallelic mutations in VPS13A, usually resulting in reduced or absent levels of its protein product, VPS13A. VPS13A localizes to contact sites between subcellular organelles, consistent with its recently identified role in lipid transfer between membranes. Mutations are associated with neuronal loss in the striatum, most prominently in the caudate nucleus, and associated marked astrogliosis. There are no other known disease-specific cellular changes (eg, protein aggregation), but autopsy reports to date have been limited, often lacking genetic or biochemical diagnostic confirmation. OBJECTIVE: The goal of this study was to characterize neuropathological findings in the brains of seven patients with VPS13A disease (chorea-acanthocytosis). METHODS: In this study, we collected brain tissues and clinical data from seven cases of VPS13A for neuropathological analysis. The clinical diagnosis was confirmed by the presence of VPS13A mutations and/or immunoblot showing the loss or reduction of VPS13A protein. Tissues underwent routine, special, and immunohistochemical staining focused on neurodegeneration. Electron microscopy was performed in one case. RESULTS: Gross examination showed severe striatal atrophy. Microscopically, there was neuronal loss and astrogliosis in affected regions. Luxol fast blue staining showed variable lipid accumulation with diverse morphology, which was further characterized by electron microscopy. In some cases, rare degenerating p62- and ubiquitin-positive cells were present in affected regions. Calcifications were present in four cases, being extensive in one. CONCLUSIONS: We present the largest autopsy series of biochemically and genetically confirmed VPS13A disease and identify novel histopathological findings implicating abnormal lipid accumulation. © 2023 International Parkinson and Movement Disorder Society.

Sphingolipid and Phospholipid Levels Are Altered in Human Brain in Chorea-Acanthocytosis.

BACKGROUND: Chorea-acanthocytosis (ChAc) is associated with mutations of VPS13A, which encodes for chorein, a protein implicated in lipid transport at intracellular membrane contact sites. OBJECTIVES: The goal of this study was to establish the lipidomic profile of patients with ChAc. METHODS: We analyzed 593 lipid species in the caudate nucleus (CN), putamen, and dorsolateral prefrontal cortex (DLPFC) from postmortem tissues of four patients with ChAc and six patients without ChAc. RESULTS: We found increased levels of bis(monoacylglycerol)phosphate, sulfatide, lysophosphatidylserine, and phosphatidylcholine ether in the CN and putamen, but not in the DLPFC, of patients with ChAc. Phosphatidylserine and monoacylglycerol were increased in the CN and N-acyl phosphatidylserine in the putamen. N-acyl serine was decreased in the CN and DLPFC, whereas lysophosphatidylinositol was decreased in the DLPFC. CONCLUSIONS: We present the first evidence of altered sphingolipid and phospholipid levels in the brains of patients with ChAc. Our observations are congruent with recent findings in cellular and animal models, and implicate defects of lipid processing in VPS13A disease pathophysiology. © 2023 International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Influence of Host Age on Intracranial AAV9 TauP301L Induced Tauopathy.

BACKGROUND: Advanced age is the greatest risk factor for the development of Alzheimer's disease (AD). This implies that some aspect of the aged milieu is possibly accelerating the development of AD related pathologies. OBJECTIVE: We hypothesized that intracranially injected with AAV9 tauP301L may cause a greater degree of pathology in old versus young mice. METHODS: Animals were injected with viral vectors overexpressing the mutant tauP301L or control protein (green fluorescent protein, GFP) into the brains of mature, middle-aged, and old C57BL/6Nia mice. The tauopathy phenotype was monitored four months after injection using behavioral, histological, and neurochemical measures. RESULTS: Phosphorylated-tau immunostaining (AT8) or Gallyas staining of aggregated tau increased with age, but other measures of tau accumulation were not significantly affected. Overall, AAV-tau injected mice had impaired radial arm water maze performance, increased microglial activation, and showed evidence of hippocampal atrophy. Aging impaired open field and rotarod performance in both AAV-tau and control mice. The efficiency of viral transduction and gene expression were the same at all animal ages. CONCLUSION: We conclude that tauP301L over expression results in a tauopathy phenotype with memory impairment and accumulation of aggregated tau. However, the effects of aging on this phenotype are modest and not detected by some markers of tau accumulation, similar to prior work on this topic. Thus, although age does influence the development of tauopathy, it is likely that other factors, such as ability to compensate for tau pathology, are more responsible for the increased risk of AD with advanced age.

Neural atrophy produced by AAV tau injections into hippocampus and anterior cortex of middle-aged mice.

Animal models of tauopathy help in understanding the role of mutations in tau pathobiology. Here, we used adeno-associated viral (AAV) vectors to administer three tau genetic variants (tauwild-type, tauP301L, and tauR406W) intracranially into 12-month-old C57BL/6Nia mice and collected tissue at 16 months. Vectors designed to express green fluorescent protein controlled for surgical procedures and exogenous protein expression by AAV. The tau genetic variants produced considerably different phenotypes. Tauwild-type and tauP301L caused memory impairments. The tauP301L caused increased amounts of aggregated tau, measured both neurochemically and histologically. Tauwild-type produced elevated levels of soluble tau and phosphorylated tau by ELISA and increased staining for phosphorylated forms of tau histologically. However, only the tauwild-type caused localized atrophy of brain tissue at the sites near the injection. The tauR406W had low protein expression and produced no atrophy or memory impairments. This supports the potential use of AAV expressing tauwild-type in aged mice to examine events leading to neurodegeneration in Alzheimer's disease pathology.

Neuropathology of Pediatric SARS-CoV-2 Infection in the Forensic Setting: Novel Application of Ex Vivo Imaging in Analysis of Brain Microvasculature.

Two years into the COVID-19 pandemic, there are few published accounts of postmortem SARS-CoV-2 pathology in children. We report 8 such cases (4 infants aged 7-36 weeks, 4 children aged 5-15 years). Four underwent ex vivo magnetic resonance neuroimaging, to assist in identification of subtle lesions related to vascular compromise. All infants were found unresponsive (3 in unsafe sleeping conditions); all but 1 had recent rhinitis and/or influenza-like illness (ILI) in the family; 1 had history of sickle cell disease. Ex vivo neuroimaging in 1 case revealed white matter (WM) signal hyperintensity and diffuse exaggeration of perivascular spaces, corresponding microscopically to WM mineralization. Neurohistology in the remaining 3 infants variably encompassed WM gliosis and mineralization; brainstem gliosis; perivascular vacuolization; perivascular lymphocytes and brainstem microglia. One had ectopic hippocampal neurons (with pathogenic variant in DEPDC5). Among the children, 3 had underlying conditions (e.g., obesity, metabolic disease, autism) and all presented with ILI. Three had laboratory testing suggesting multisystem inflammatory syndrome (MIS-C). Two were hospitalized for critical care including mechanical ventilation and extracorporeal membrane oxygenation (ECMO); one (co-infected with adenovirus) developed right carotid stroke ipsilateral to the ECMO cannula and the other required surgery for an ingested foreign body. Autopsy findings included: acute lung injury in 3 (1 with microthrombi); and one each with diabetic ketoacidosis and cardiac hypertrophy; coronary and cerebral arteritis and aortitis, resembling Kawasaki disease; and neuronal storage and enlarged fatty liver. All 4 children had subtle meningoencephalitis, focally involving the brainstem. On ex vivo neuroimaging, 1 had focal pontine susceptibility with corresponding perivascular inflammation/expanded perivascular spaces on histopathology. Results suggest SARS-CoV-2 in infants may present as sudden unexpected infant death, while in older children, signs and symptoms point to severe disease. Underlying conditions may predispose to fatal outcomes. As in adults, the neuropathologic changes may be subtle, with vascular changes such as perivascular vacuolization and gliosis alongside sparse perivascular lymphocytes. Detection of subtle vascular pathology is enhanced by ex vivo neuroimaging. Additional analysis of the peripheral/autonomic nervous system and investigation of co-infection in children with COVID-19 is necessary to understand risk for cardiovascular collapse/sudden death.

Is Memory Decline Associated With Inflammatory Response?

OBJECTIVE: To examine whether changes in memory over a 10-year period could predict a change in C-reactive protein (CRP) levels. METHOD: A mixed model analysis was first conducted to obtain the estimates for change in memory over the 10-year period using data from the Health and Retirement Study. Then a multivariate regression to determine whether a change in episodic memory could predict subsequent CRP levels was conducted. Furthermore, a general linear model was conducted to determine differences in CRP levels among different rates of change in episodic memory. RESULTS: Greater declines in episodic memory were associated with higher levels of subsequent CRP (Estimate = -0.32, SE = 0.12, ß = -.03, p = .008). The general linear model revealed that those with greater memory declines were more likely to have higher levels of CRP, F = 26.50, p < .001. DISCUSSION: These results highlight the notion that memory decline and inflammation may be intertwined, and we discuss various avenues that warrant further investigation.

Systematic review and meta-analyses of useful field of view cognitive training.

Systematic review and meta-analyses were conducted of Useful Field of View (UFOV) training, which was evaluated by Institute of Medicine criteria. Forty-four studies of UFOV training from 17 randomized trials conducted among adults were identified in systematic review. Results addressing the Institute of Medicine criteria indicated that: (a) UFOV training enhanced neural outcomes, speed of processing, and attention. (b) UFOV training effects were equivalent when compared to active- or no-contact control conditions. (c) UFOV training showed far transfer to everyday function. (d) Improvements on the trained skills endured across ten years. (e) Half of the clinical trials identified were conducted by researchers without financial interests in UFOV training. Results indicated that UFOV training effects were larger for adaptive- than non-adaptive training techniques, and in community-based as compared to clinical samples. UFOV training did not transfer to other neuropsychological outcomes, but positively enhanced well-being, health, and quality of life longitudinally. Criticisms of cognitive training are addressed. UFOV training should be implemented among older adults to improve real-world functional outcomes and well-being.

Auditory Processing of Older Adults With Probable Mild Cognitive Impairment.

Purpose: Studies suggest that deficits in auditory processing predict cognitive decline and dementia, but those studies included limited measures of auditory processing. The purpose of this study was to compare older adults with and without probable mild cognitive impairment (MCI) across two domains of auditory processing (auditory performance in competing acoustic signals and temporal aspects of audition). Method: The Montreal Cognitive Assessment (Nasreddine et al., 2005) was used to classify participants as with or without probable MCI. In this cross-sectional study, participants (n = 79) completed 4 measures of auditory processing: Synthetic Sentence Identification with Ipsilateral Competing Message (Gates, Beiser, Rees, D'Agostino, & Wolf, 2002), Dichotic Sentence Identification (Fifer, Jerger, Berlin, Tobey, & Campbell, 1983), Adaptive Tests of Temporal Resolution (ATTR; Lister & Roberts, 2006; across-channel and within-channel subtests), and time-compressed speech (Wilson, 1993; Wilson, Preece, Salamon, Sperry, & Bornstein, 1994). Audiometry was also conducted. Results: Those with probable MCI had significantly poorer performance than those without MCI on Synthetic Sentence Identification with Ipsilateral Competing Message, Dichotic Sentence Identification, and the ATTR within-channel subtest. No group differences were found for time-compressed speech, ATTR across-channel, or audiometric measures. Conclusions: Older adults with cognitive impairment not only have difficulty with competing acoustic signals but may also show poor temporal processing. The profile of auditory processing deficits among older adults with cognitive impairment may include multiple domains.