Nanobodies (Nbs), the single-domain antigen-binding fragments of dromedary Heavy-chain antibodies (HCAb), are excellent candidates as therapeutic and diagnostic tools in synucleinopathies because of their small size, solubility and stability. Here, we constructed an immune nanobody library specific to the monomeric form of alpha-synuclein (α-syn). Phage display screening of the library allowed the identification of a nanobody, Nbα-syn01, specific for α-syn. Unlike previously developed nanobodies, Nbα-syn01 recognized the N-terminal region which is critical for in vitro and in vivo aggregation and contains many point mutations involved in early PD cases. The affinity of the monovalent Nbα-syn01 and the engineered bivalent format BivNbα-syn01 measured by Isothermal titration calorimetry revealed unexpected results where Nbα-syn01 and its bivalent format recognized preferentially α-syn fibrils compared to the monomeric form. Nbα-syn01 and BivNbα-syn01 were also able to inhibit α-syn-seeded aggregation in vitro and reduced α-syn-seeded aggregation and toxicity in cells showing their potential to reduce α-syn pathology. Moreover, both nanobody formats were able to recognize Lewy-body pathology in human post-mortem brain tissue from PD and DLB cases. Additionally, we present evidence through structural docking that Nbα-syn01 binds the N-terminal region of the α-syn aggregated form. Overall, these results highlight the potential of Nbα-syn01 and BivNbα-syn01 in developing into a diagnostic or a therapeutic tool for PD and related disorders.

Cancers represent complex autonomous systems, displaying self‐sufficiency in growth signaling. Autonomous growth is fueled by a cancer cell's ability to “secrete‐and‐sense” growth factors (GFs): a poorly understood phenomenon. Using an integrated computational and experimental approach, here we dissect the impact of a feedback‐coupled GTPase circuit within the secretory pathway that imparts secretion‐coupled autonomy. The circuit is assembled when the Ras‐superfamily monomeric GTPase Arf1, and the heterotrimeric GTPase Giαβγ and their corresponding GAPs and GEFs are coupled by GIV/Girdin, a protein that is known to fuel aggressive traits in diverse cancers. One forward and two key negative feedback loops within the circuit create closed‐loop control, allow the two GTPases to coregulate each other, and convert the expected switch‐like behavior of Arf1‐dependent secretion into an unexpected dose–response alignment behavior of sensing and secretion. Such behavior translates into cell survival that is self‐sustained by stimulus‐proportionate secretion. Proteomic studies and protein–protein interaction network analyses pinpoint GFs (e.g., the epidermal GF) as key stimuli for such self‐sustenance. Findings highlight how the enhanced coupling of two biological switches in cancer cells is critical for multiscale feedback control to achieve secretion‐coupled autonomy of growth factors.

Synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of α-synuclein (α-syn). Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, antibody-based immunotherapy holds much promise. However, the large size of antibodies and corresponding difficulty in crossing the blood-brain barrier has limited development in this area. To overcome this issue, we engineered single-chain variable fragments (scFvs) against fibrillar α-syn, a putative disease-relevant form of α-syn. The purified scFvs showed specific activity towards α-syn fibrils and oligomers in comparison to monomers and recognized intracellular inclusions in human post-mortem brain tissue of Lewy body disease cases, but not aged controls. In vitro studies indicated scFvs inhibit the seeding of α-syn aggregation in a time-dependent manner, decreased α-syn seed-induced toxicity in a cell model of PD, and reduced the production of insoluble α-syn phosphorylated at Ser-129 (pS129-α-syn). These results suggest that our α-syn fibril-specific scFvs recognize α-syn pathology and can inhibit the aggregation of α-syn in vitro and prevent seeding-dependent toxicity. Therefore, the scFvs described here have considerable potential to be utilized towards immunotherapy in synucleinopathies and may also have applications in ante-mortem imaging modalities.

The aggregation of α-synuclein (α-syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of synucleinopatheis, including Parkinson’s disease (PD). A further characteristic of PD is the oxidative stress that results in the formation of aldehydes by lipid peroxidation. It has been reported that the brains of deceased patients with PD contain high levels of protein oligomers that are cross-linked to these aldehydes. Increasing evidence also suggests that prefibrillar oligomeric species are more toxic than the mature amyloid fibrils. However, due to the heterogenous and metastable nature, characterization of the α-syn oligomeric species has been challenging. Here, we generated and characterized distinct α-syn oligomers in vitro in the presence of DA and lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE oligomer were stable towards the treatment with SDS, urea, and temperature. The secondary structure analysis revealed that only HNE and ONE oligomers contain β-sheet content. In the seeding assay, both DA and ONE oligomers significantly accelerated the aggregation. Furthermore, all oligomeric preparations were found to seed the aggregation of α-syn monomers in vitro and found to be cytotoxic when added to SH-SY5Y cells. Finally, both HNE and ONE α-syn oligomers can be used as a calibrator in an α-syn oligomers-specific ELISA.

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by deficits in social communication, restricted interests, and repetitive behaviors. Despite considerable research efforts, the genetic complexity of ASD remains poorly understood, complicating diagnosis and treatment, especially in the Arab population, with its genetic diversity linked to migration, tribal structures, and high consanguinity. To address the scarcity of ASD genetic data in the Middle East, we conducted genome sequencing (GS) on 50 ASD subjects and their unaffected parents. Our analysis revealed 37 single-nucleotide variants from 36 candidate genes and over 200 CGG repeats in the FMR1 gene in one subject. The identified variants were classified as uncertain, likely pathogenic, or pathogenic based on in-silico algorithms and ACMG criteria. Notably, 52% of the identified variants were homozygous, indicating a recessive genetic architecture to ASD in this population. This finding underscores the significant impact of high consanguinity within the Qatari population, which could be utilized in genetic counseling/screening program in Qatar. We also discovered single nucleotide variants in 13 novel genes not previously associated with ASD: ARSF, BAHD1, CHST7, CUL2, FRMPD3, KCNC4, LFNG, RGS4, RNF133, SCRN2, SLC12A8, USP24, and ZNF746. Our investigation categorized the candidate genes into seven groups, highlighting their roles in cognitive development, including the ubiquitin pathway, transcription factors, solute carriers, kinases, glutamate receptors, chromatin remodelers, and ion channels.