ENGINEERED NANOBODIES WITH PROGRAMMABLE TARGET ANTIGEN PROTEOLYSIS (PTAP) FUSIONS REGULATE INTRACELLULAR ALPHA-SYNUCLEIN IN VITRO AND IN VIVO.

Alpha-synuclein (αSyn) aggregation and the formation of Lewy pathology (LP) is a foundational pathophysiological phenomenon in synucleinopathies. Delivering therapeutic single-chain and single-domain antibodies that bind pathogenic targets can disrupt intracellular aggregation. The fusion of antibody fragments to a negatively-charged proteasomal targeting motif (PEST) creates bifunctional constructs that enhance both solubility and turnover. With sequence-specific point mutations of PEST sequences that modulate proteasomal degradation efficiency, we report the creation of Programmable Target Antigen Proteolysis (PTAP) technology that can provide graded control over the levels of target antigens. We have previously demonstrated our lead anti-αSyn intrabody, VH14-PEST, is capable of reducing the pathological burden of synucleinopathy in vitro and in vivo. Here, we report a family of fully humanized VH14-PTAP constructs for controllable, therapeutic targeting of intracellular α-Syn. In cells, we demonstrate successful target engagement and efficacy of VH14-hPEST intrabodies, and validate proof-of-principle in human cells using 3D human organoids derived from PD-patient induced pluripotent stem cells (iPSC). In two synuclein-based rat models, PTAP intrabodies attenuated nigral αSyn pathology, preserved nigrostriatal dopaminergic tone, and slowed the propagation of αSyn pathology. These data demonstrate the potency of intracellular αSyn targeting as a method to alleviate pathology and highlight the potential clinical utility of PTAP intrabodies.

Mechanisms of Glucocerebrosidase Dysfunction in Parkinson's Disease.

Beta-glucocerebrosidase is a lysosomal hydrolase, encoded by GBA1 that represents the most common risk gene associated with Parkinson's disease (PD) and Lewy Body Dementia. Glucocerebrosidase dysfunction has been also observed in the absence of GBA1 mutations across different genetic and sporadic forms of PD and related disorders, suggesting a broader role of glucocerebrosidase in neurodegeneration. In this review, we highlight recent advances in mechanistic characterization of glucocerebrosidase function as the foundation for development of novel therapeutics targeting glucocerebrosidase in PD and related disorders.

In situ proximity labeling identifies Lewy pathology molecular interactions in the human brain.

The intracellular misfolding and accumulation of alpha-synuclein into structures collectively called Lewy pathology (LP) is a central phenomenon for the pathogenesis of synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Understanding the molecular architecture of LP is crucial for understanding synucleinopathy disease origins and progression. Here we used a technique called biotinylation by antibody recognition (BAR) to label total (BAR-SYN1) and pathological alpha-synuclein (BAR-PSER129) in situ for subsequent mass spectrometry analysis. Results showed superior immunohistochemical detection of LP following the BAR-PSER129 protocol, particularly for fibers and punctate pathology within the striatum and cortex. Mass spectrometry analysis of BAR-PSER129-labeled LP identified 261 significantly enriched proteins in the synucleinopathy brain when compared to nonsynucleinopathy brains. In contrast, BAR-SYN1 did not differentiate between disease and nonsynucleinopathy brains. Pathway analysis of BAR-PSER129-enriched proteins revealed enrichment for 718 pathways; notably, the most significant KEGG pathway was PD, and Gene Ontology (GO) cellular compartments were the vesicle, extracellular vesicle, extracellular exosome, and extracellular organelle. Pathway clustering revealed several superpathways, including metabolism, mitochondria, lysosome, and intracellular vesicle transport. Validation of the BAR-PSER129-identified protein hemoglobin beta (HBB) by immunohistochemistry confirmed the interaction of HBB with PSER129 Lewy neurites and Lewy bodies. In summary, BAR can be used to enrich for LP from formalin-fixed human primary tissues, which allowed the determination of molecular signatures of LP. This technique has broad potential to help understand the phenomenon of LP in primary human tissue and animal models.

Enhanced CNS transduction from AAV.PHP.eB infusion into the cisterna magna of older adult rats compared to AAV9.

The development of high efficiency, central nervous system (CNS) targeting AAV-based gene therapies is necessary to address challenges in both pre-clinical and clinical investigations. The engineered capsids, AAV.PHP.B and AAV.PHP.eB, show vastly improved blood-brain barrier penetration compared to their parent serotype, AAV9, but with variable effect depending on animal system, strain, and delivery route. As most characterizations of AAV.PHP variants have been performed in mice, it is currently unknown whether AAV.PHP variants improve CNS targeting when delivered intrathecally in rats. We evaluated the comparative transduction efficiencies of equititer doses (6 × 1011vg) of AAV.PHP.eB-CAG-GFP and AAV9-CAG-GFP when delivered into the cisterna magna of 6-9-month old rats. Using both quantitative and qualitative assessments, we observed consistently superior biodistribution of GFP+ cells and fibers in animals treated with AAV.PHP.eB compared to those treated with AAV9. Enhanced GFP signal was uniformly observed throughout rostrocaudal brain regions in AAV.PHP.eB-treated animals with matching GFP protein expression detected in the forebrain, midbrain, and cerebellum. Collectively, these data illustrate the benefit of intracisternal infusions of AAV.PHP.eB as an optimal system to distribute CNS gene therapies in preclinical investigations of rats, and may have important translational implications for the clinical CNS targeting.

Viral-based rodent and nonhuman primate models of multiple system atrophy: Fidelity to the human disease.

Multiple system atrophy (MSA) is a rare and extremely debilitating progressive neurodegenerative disease characterized by variable combinations of parkinsonism, cerebellar ataxia, dysautonomia, and pyramidal dysfunction. MSA is a unique synucleinopathy, in which alpha synuclein-rich aggregates are present in the cytoplasm of oligodendroglia. The precise origin of the alpha synuclein (aSyn) found in the glial cytoplasmic inclusions (GCIs) as well the mechanisms of neurodegeneration in MSA remain unclear. Despite this fact, cell and animal models of MSA rely on oligodendroglial overexpression of aSyn. In the present study, we utilized a novel oligotrophic AAV, Olig001, to overexpress aSyn specifically in striatal oligodendrocytes of rats and nonhuman primates in an effort to further characterize our novel viral vector-mediated MSA animal models. Using two cohorts of animals with 10-fold differences in Olig001 vector titers, we show a dose-dependent formation of MSA-like pathology in rats. High titer of Olig001-aSyn in these animals were required to produce the formation of pS129+ and proteinase K resistant aSyn-rich GCIs, demyelination, and neurodegeneration. Using this knowledge, we injected high titer Olig001 in the putamen of cynomolgus macaques. After six months, histological analysis showed that oligodendroglial overexpression of aSyn resulted in the formation of hallmark GCIs throughout the putamen, demyelination, a 44% reduction of striatal neurons and a 12% loss of nigral neurons. Furthermore, a robust inflammatory response similar to MSA was produced in Olig001-aSyn NHPs, including microglial activation, astrogliosis, and a robust infiltration of T cells into the CNS. Taken together, oligodendroglial-specific viral vector-mediated overexpression of aSyn in rats and nonhuman primates faithfully reproduces many of the pathological disease hallmarks found in MSA. Future studies utilizing these large animal models of MSA would prove extremely valuable as a pre-clinical platform to test novel therapeutics that are so desperately needed for MSA.

Immunotherapy in Parkinson's disease: Current status and future directions.

Immunotherapeutic approaches for the treatment of Parkinson's disease (PD) and related synucleinopathies have steadily developed over the last two decades with several iterations currently being tested in clinical trials. Although classically characterized as a movement disorder, PD is also defined clinically by numerous non-motor features that can precede the motor manifestations and span across several decades of disease progression. Pathologically, PD is characterized by proteinaceous inclusions that largely consist of misfolded and aggregated forms of the protein, alpha-synuclein. Recent research has proposed that alpha-synuclein pathology is capable of propagating from cell-to-cell, and thus, may cause the clinical progression of the disease. Antibody-based therapies are ideal drugs to theoretically target pathological proteins, especially those located in the extracellular space. Several other targeting strategies have been developed to indirectly mitigate the propagation of alpha-synuclein and are in various stages of pre-clinical and clinical development. In this review, we discuss the current status of development for immunotherapies in PD as well as the primary challenges that must be hurdled to bring an effective immunotherapeutic drug to market.

Loss of One Engrailed1 Allele Enhances Induced α-Synucleinopathy.

BACKGROUND: Parkinson's disease (PD) is a synucleinopathy that has multiple neuropathological characteristics, with nigrostriatal dopamine system degeneration being a core feature. Current models of PD pathology typically fail to recapitulate several attributes of the pathogenic process and neuropathology. We aimed to define the effects of combining a mouse model exhibiting multiple PD-like changes with intrastriatal injections of α-synuclein (α-syn) pre-formed fibril (PFFs) aggregates. We employed the heterozygous Engrailed 1 (En1+/-) mouse that features several pathophysiological hallmarks of clinical PD. OBJECTIVE: To test the hypothesis that the neuropathological changes in the En1+/- mice will promote formation of α-syn aggregates following intrastriatal injections of pathogenic human α-syn PFFs. METHODS: We unilaterally injected PFFs into the striata of 1-month-old En1+/- and control wild-type mice and euthanized animals at 3 months for post-mortem analysis. RESULTS: Using immunohistochemistry and unbiased stereology, we established that PFF-injected En1+/- mice exhibited a near-threefold increase in pS129-α-syn-positive neurons in the substantia nigra compared to PFF-injected wild-type mice. The PFF-injected En1+/- mice also displayed significant increases in pS129-α-syn-positive neurons in the amygdala and ventral tegmental area; regions of known PD pathology with projections to the striatum. Additionally, we observed amplified pS129-α-syn-positive aggregation in En1+/- mice in multiple cortical regions. CONCLUSIONS: Following intrastriatal injection of PFFs, absence of an En1 allele leads to additional aggregation of pathological α-syn, potentially due to En1-loss mediated nigrostriatal impairment. We propose that further development of this double-hit model could result in a PD mouse model that predicts which experimental therapies will be effective in PD.

Proteasome-targeted nanobodies alleviate pathology and functional decline in an α-synuclein-based Parkinson's disease model.

Therapeutics designed to target α-synuclein (α-syn) aggregation may be critical in halting the progression of pathology in Parkinson's disease (PD) patients. Nanobodies are single-domain antibody fragments that bind with antibody specificity, but allow readier genetic engineering and delivery. When expressed intracellularly as intrabodies, anti-α-syn nanobodies fused to a proteasome-targeting proline, aspartate or glutamate, serine, and threonine (PEST) motif can modulate monomeric concentrations of target proteins. Here we aimed to validate and compare the in vivo therapeutic potential of gene therapy delivery of two proteasome-directed nanobodies selectively targeting α-syn in a synuclein overexpression-based PD model: VH14*PEST (non-amyloid component region) and NbSyn87*PEST (C-terminal region). Stereotaxic injections of adeno-associated viral 5-α-syn (AAV5-α-syn) into the substantia nigra (SN) were performed in Sprague-Dawley rats that were sorted into three cohorts based on pre-operative behavioral testing. Rats were treated with unilateral SN injections of vectors for VH14*PEST, NbSyn87*PEST, or injected with saline 3 weeks post lesion. Post-mortem assessments of the SN showed that both nanobodies markedly reduced the level of phosphorylated Serine-129 α-syn labeling relative to saline-treated animals. VH14*PEST showed considerable maintenance of striatal dopaminergic tone in comparison to saline-treated and NbSyn87*PEST-treated animals as measured by tyrosine hydroxylase immunoreactivity (optical density), DAT immunoreactivity (optical density), and dopamine concentration (high-performance liquid chromatography). Microglial accumulation and inflammatory response, assessed by stereological counts of Iba-1-labeled cells, was modestly increased in NbSyn87*PEST-injected rats but not in VH14*PEST-treated or saline-treated animals. Modest behavioral rescue was also observed, although there was pronounced variability among individual animals. These data validate in vivo therapeutic efficacy of vector-delivered intracellular nanobodies targeting α-syn misfolding and aggregation in synucleinopathies such as PD.

Hypoxia/reoxygenation impairs memory formation via adenosine-dependent activation of caspase 1.

After hypoxia, a critical adverse outcome is the inability to create new memories. How anterograde amnesia develops or resolves remains elusive, but a link to brain-based IL-1 is suggested due to the vital role of IL-1 in both learning and brain injury. We examined memory formation in mice exposed to acute hypoxia. After reoxygenation, memory recall recovered faster than memory formation, impacting novel object recognition and cued fear conditioning but not spatially cued Y-maze performance. The ability of mice to form new memories after hypoxia/reoxygenation was accelerated in IL-1 receptor 1 knockout (IL-1R1 KO) mice, in mice receiving IL-1 receptor antagonist (IL-1RA), and in mice given the caspase 1 inhibitor Ac-YVAD-CMK. Mechanistically, hypoxia/reoxygenation more than doubled caspase 1 activity in the brain, which was localized to the amygdala compared to the hippocampus. This reoxygenation-dependent activation of caspase 1 was prevented by broad-spectrum adenosine receptor (AR) antagonism with caffeine and by targeted A1/A2A AR antagonism with 8-cyclopentyl-1,3-dipropylxanthine plus 3,7-dimethyl-1-propargylxanthine. Additionally, perfusion of adenosine activated caspase 1 in the brain, while caffeine blocked this action by adenosine. Finally, resolution of anterograde amnesia was improved by both caffeine and by targeted A1/A2A AR antagonism. These findings indicate that amygdala-based anterograde amnesia after hypoxia/reoxygenation is sustained by IL-1ß generated through adenosine-dependent activation of caspase 1 after reoxygenation.