Targeted brain-specific tauopathy compromises peripheral skeletal muscle integrity and function.

Tauopathies are neurodegenerative disorders in which the pathological intracellular aggregation of the protein tau causes cognitive deficits. Additionally, clinical studies report muscle weakness in populations with tauopathy. However, whether neuronal pathological tau species confer muscle weakness, and whether skeletal muscle maintains contractile capacity in primary tauopathy remains unknown. Here, we identified skeletal muscle abnormalities in a mouse model of primary tauopathy, expressing human mutant P301L-tau using adeno-associated virus serotype 8 (AAV8). AAV8-P301L mice showed grip strength deficits, hyperactivity, and abnormal histological features of skeletal muscle. Additionally, spatially resolved gene expression of muscle cross sections were altered in AAV8-P301L myofibers. Transcriptional changes showed alterations of genes encoding sarcomeric proteins, proposing a weakness phenotype. Strikingly, specific force of the soleus muscle was blunted in AAV8-P301L tau male mice. Our findings suggest tauopathy has peripheral consequences in skeletal muscle that contribute to weakness in tauopathy.

Aß Amyloid Scaffolds the Accumulation of Matrisome and Additional Proteins in Alzheimer's Disease.

We report a highly significant correlation in brain proteome changes between Alzheimers disease (AD) and CRND8 APP695NL/F transgenic mice. However, integrating protein changes observed in the CRND8 mice with co-expression networks derived from human AD, reveals both conserved and divergent module changes. For the most highly conserved module (M42, matrisome) we find many proteins accumulate in plaques, cerebrovascular amyloid (CAA), dystrophic processes, or a combination thereof. Overexpression of two M42 proteins, midkine (Mdk) and pleiotrophin (PTN), in CRND8 mice brains leads to increased accumulation of A ß ; in plaques and in CAA; further, recombinant MDK and PTN enhance A ß ; aggregation into amyloid. Multiple M42 proteins, annotated as heparan sulfate binding proteins, bind to fibrillar A ß 42 and a non-human amyloid fibril in vitro. Supporting this binding data, MDK and PTN co-accumulate with transthyretin (TTR) amyloid in the heart and islet amyloid polypeptide (IAPP) amyloid in the pancreas. Our findings establish several critical insights. Proteomic changes in modules observed in human AD brains define an A ß ; amyloid responsome that is well conserved from mouse model to human. Further, distinct amyloid structures may serve as scaffolds, facilitating the co-accumulation of proteins with signaling functions. We hypothesize that this co-accumulation may contribute to downstream pathological sequalae. Overall, this contextualized understanding of proteomic changes and their interplay with amyloid deposition provides valuable insights into the complexity of AD pathogenesis and potential biomarkers and therapeutic targets.

Targeted brain-specific tauopathy compromises peripheral skeletal muscle integrity and function.

Tauopathies are neurodegenerative disorders in which the pathological intracellular aggregation of the protein tau causes cognitive deficits. Additionally, clinical studies report muscle weakness in populations with tauopathy. However, whether neuronal pathological tau species confer muscle weakness, and whether skeletal muscle maintains contractile capacity in primary tauopathy remains unknown. Here, we identified skeletal muscle abnormalities in a mouse model of primary tauopathy, expressing human mutant P301L-tau using adeno-associated virus serotype 8 (AAV8). AAV8-P301L mice showed grip strength deficits, hyperactivity, and abnormal histological features of skeletal muscle. Additionally, spatially resolved gene expression of muscle cross sections were altered in AAV8-P301L myofibers. Transcriptional changes showed alterations of genes encoding sarcomeric proteins, proposing a weakness phenotype. Strikingly, specific force of the soleus muscle was blunted in AAV8-P301L tau male mice. Our findings suggest tauopathy has peripheral consequences in skeletal muscle that contribute to weakness in tauopathy.

A C1qTNF3 collagen domain fusion chaperones diverse secreted proteins and anti-Aß scFvs: Applications for gene therapies.

Enhancing production of protein cargoes delivered by gene therapies can improve efficacy by reducing the amount of vector or simply increasing transgene expression levels. We explored the utility of a 126-amino acid collagen domain (CD) derived from the C1qTNF3 protein as a fusion partner to chaperone secreted proteins, extracellular "decoy receptor" domains, and single-chain variable fragments (scFvs). Fusions to the CD domain result in multimerization and enhanced levels of secretion of numerous fusion proteins while maintaining functionality. Efficient creation of bifunctional proteins using the CD domain is also demonstrated. Recombinant adeno-associated viral vector delivery of the CD with a signal peptide resulted in high-level expression with minimal biological impact as assessed by whole-brain transcriptomics. As a proof-of-concept in vivo study, we evaluated three different anti-amyloid Aß scFvs (anti-Aß scFvs), alone or expressed as CD fusions, following viral delivery to neonatal CRND8 mice. The CD fusion increased half-life, expression levels, and improved efficacy for amyloid lowering of a weaker binding anti-Aß scFv. These studies validate the potential utility of this small CD as a fusion partner for secretory cargoes delivered by gene therapy and demonstrate that it is feasible to use this CD fusion to create biotherapeutic molecules with enhanced avidity or bifunctionality.

Deletion of Abi3/Gngt2 influences age-progressive amyloid ß and tau pathologies in distinctive ways.

BACKGROUND: The S209F variant of Abelson Interactor Protein 3 (ABI3) increases risk for Alzheimer's disease (AD), but little is known about its function in relation to AD pathogenesis. METHODS: Here, we use a mouse model that is deficient in Abi3 locus to study how the loss of function of Abi3 impacts two cardinal neuropathological hallmarks of AD-amyloid ß plaques and tau pathology. Our study employs extensive neuropathological and transcriptomic characterization using transgenic mouse models and adeno-associated virus-mediated gene targeting strategies. RESULTS: Analysis of bulk RNAseq data confirmed age-progressive increase in Abi3 levels in rodent models of AD-type amyloidosis and upregulation in AD patients relative to healthy controls. Using RNAscope in situ hybridization, we localized the cellular distribution of Abi3 in mouse and human brains, finding that Abi3 is expressed in both microglial and non-microglial cells. Next, we evaluated Abi3-/- mice and document that both Abi3 and its overlapping gene, Gngt2, are disrupted in these mice. Using multiple transcriptomic datasets, we show that expression of Abi3 and Gngt2 are tightly correlated in rodent models of AD and human brains, suggesting a tight co-expression relationship. RNAseq of the Abi3-Gngt2-/- mice revealed upregulation of Trem2, Plcg2, and Tyrobp, concomitant with induction of an AD-associated neurodegenerative signature, even in the absence of AD-typical neuropathology. In APP mice, loss of Abi3-Gngt2 resulted in a gene dose- and age-dependent reduction in Aß deposition. Additionally, in Abi3-Gngt2-/- mice, expression of a pro-aggregant form of human tau exacerbated tauopathy and astrocytosis. Further, using in vitro culture assays, we show that the AD-associated S209F mutation alters the extent of ABI3 phosphorylation. CONCLUSIONS: These data provide an important experimental framework for understanding the role of Abi3-Gngt2 function and early inflammatory gliosis in AD. Our studies also demonstrate that inflammatory gliosis could have opposing effects on amyloid and tau pathology, highlighting the unpredictability of targeting immune pathways in AD.

Interleukin-12/23 deficiency differentially affects pathology in male and female Alzheimer's disease-like mice.

Pathological aggregation of amyloid-ß (Aß) is a main hallmark of Alzheimer's disease (AD). Recent genetic association studies have linked innate immune system actions to AD development, and current evidence suggests profound gender differences in AD pathogenesis. Here, we characterise gender-specific pathologies in the APP23 AD-like mouse model and find that female mice show stronger amyloidosis and astrogliosis compared with male mice. We tested the gender-specific effect of lack of IL12p40, the shared subunit of interleukin (IL)-12 and IL-23, that we previously reported to ameliorate pathology in APPPS1 mice. IL12p40 deficiency gender specifically reduces Aß plaque burden in male APP23 mice, while in female mice, a significant reduction in soluble Aß1-40 without changes in Aß plaque burden is seen. Similarly, plasma and brain cytokine levels are altered differently in female versus male APP23 mice lacking IL12p40, while glial properties are unchanged. These data corroborate the therapeutic potential of targeting IL-12/IL-23 signalling in AD, but also highlight the importance of gender considerations when studying the role of the immune system and AD.

A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity.

Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.

Nuclear translocation uncovers the amyloid peptide Aß42 as a regulator of gene transcription.

Although soluble species of the amyloid-ß peptide Aß42 correlate with disease symptoms in Alzheimer disease, little is known about the biological activities of amyloid-ß (Aß). Here, we show that Aß peptides varying in lengths from 38 to 43 amino acids are internalized by cultured neuroblastoma cells and can be found in the nucleus. By three independent methods, we demonstrate direct detection of nuclear Aß42 as follows: (i) biochemical analysis of nuclear fractions; (ii) detection of biotin-labeled Aß in living cells by confocal laser scanning microscopy; and (iii) transmission electron microscopy of Aß in cultured cells, as well as brain tissue of wild-type and transgenic APPPS1 mice (overexpression of amyloid precursor protein and presenilin 1 with Swedish and L166P mutations, respectively). Also, this study details a novel role for Aß42 in nuclear signaling, distinct from the amyloid precursor protein intracellular domain. Chromatin immunoprecipitation showed that Aß42 specifically interacts as a repressor of gene transcription with LRP1 and KAI1 promoters. By quantitative RT-PCR, we confirmed that mRNA levels of the examined candidate genes were exclusively decreased by the potentially neurotoxic Aß42 wild-type peptide. Shorter peptides (Aß38 or Aß40) and other longer peptides (nontoxic Aß42 G33A substitution or Aß43) did not affect mRNA levels. Overall, our data indicate that the nuclear translocation of Aß42 impacts gene regulation, and deleterious effects of Aß42 in Alzheimer disease pathogenesis may be influenced by altering the expression profiles of disease-modifying genes.

Inhibition of IL-12/IL-23 signaling reduces Alzheimer's disease-like pathology and cognitive decline.

The pathology of Alzheimer's disease has an inflammatory component that is characterized by upregulation of proinflammatory cytokines, particularly in response to amyloid-ß (Aß). Using the APPPS1 Alzheimer's disease mouse model, we found increased production of the common interleukin-12 (IL-12) and IL-23 subunit p40 by microglia. Genetic ablation of the IL-12/IL-23 signaling molecules p40, p35 or p19, in which deficiency of p40 or its receptor complex had the strongest effect, resulted in decreased cerebral amyloid load. Although deletion of IL-12/IL-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in a reduction of cerebral amyloid load in APPPS1 mice. Furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced the concentration of soluble Aß species and reversed cognitive deficits in aged APPPS1 mice. The concentration of p40 was also increased in the cerebrospinal fluid of subjects with Alzheimer's disease, which suggests that inhibition of the IL-12/IL-23 pathway may attenuate Alzheimer's disease pathology and cognitive deficits.

M2 polarized macrophages and giant cells contribute to myofibrosis in neuromuscular sarcoidosis.

The etiopathogenesis of sarcoidosis, a systemic granulomatous disease, still remains obscure. A multitude of organs have been described to be affected in systemic sarcoidosis. Skeletal muscles may also be affected, leading to myalgia and weakness. A workup of the specific immune response with emphasis on the macrophage response is provided herein. Affected muscle tissue from seven patients with systemic sarcoidosis was analyzed and compared with that from seven patients with other myopathies containing macrophagocytic infiltration. Monocytes/macrophages and giant cells in granulomas of muscle tissue from patients with sarcoidosis show a status of alternative activation (M2) based on their expression of CD206, CD301, arginase-1, and suppressor of cytokine signaling-1 as a consequence of a functionally type 2 helper T cell (Th2)-biased cytokine profile. Significant fibrosis and up-regulation of CCL18 were associated with the M2 phenotype of macrophages. Conversely, up-regulated Th1 cytokines did not result in significant classical activation of macrophages (M1), with poor inducible nitric oxide synthase and cyclooxygenase-2 production. Giant cell formation was further associated with up-regulated expression of DNAX-activating protein of 12 kDa (DAP12; gene symbol TYROBP). Functionally, alternative activation of macrophages on the basis of a Th2-biased immune response may induce clinical symptoms and chronic evolution of neuromuscular sarcoidosis. This is the first characterization of Th2-mediated immune mechanisms in neuromuscular sarcoidosis suggesting that a specific macrophage activation status leading to myofibrosis may be a key event in the pathogenesis of this disease.

Fetal akinesia caused by a novel actin filament aggregate myopathy skeletal muscle actin gene (ACTA1) mutation.

We report a female newborn, diagnosed with fetal akinesia in utero, who died one hour after birth. Post-mortem muscle biopsy demonstrated actin-filament myopathy based on immunolabelling for sarcomeric actin, and large areas of filaments, without rod formation, ultrastructurally. Analysis of DNA extracted from the muscle disclosed a novel de novo heterozygous c.44G>A, GGC>GAC, 'p.Gly15Asp' mutation in the ACTA1 gene. Analysis of the location of the mutated amino-acid in the actin molecule suggests the mutation most likely causes abnormal nucleotide binding, and consequent pathological actin polymerization. This case emphasizes the association of fetal akinesia with actin-filament myopathy.

Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses.

Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying cerebral amyloidoses. Here we report the chemical design of pentameric thiophene derivatives, denoted luminescent conjugated oligothiophenes (LCOs), which could be used for real-time visualization of cerebral protein aggregates in transgenic mouse models of neurodegenerative diseases by multiphoton microscopy. One of the LCOs, p-FTAA, could be utilized for ex vivo spectral assignment of distinct prion deposits from two mouse-adapted prion strains. p-FTAA also revealed staining of transient soluble pre-fibrillar non-thioflavinophilic Abeta-assemblies during in vitro fibrillation of Abeta peptides. In brain tissue samples, Abeta deposits and neurofibrillary tangles (NFTs) were readily identified by a strong fluorescence from p-FTAA and the LCO staining showed complete co-localization with conventional antibodies (6E10 and AT8). In addition, a patchy islet-like staining of individual Abeta plaque was unveiled by the anti-oligomer A11 antibody during co-staining with p-FTAA. The major hallmarks of Alzheimer's disease, namely, Abeta aggregates versus NFTs, could also be distinguished because of distinct emission spectra from p-FTAA. Overall, we demonstrate that LCOs can be utilized as powerful practical research tools for studying protein aggregation diseases and facilitate the study of amyloid origin, evolution and maturation, Abeta-tau interactions, and pathogenesis both ex vivo and in vivo.