Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases.

The abnormal aggregation and accumulation of alpha-synuclein (aSyn) in the brain is a defining hallmark of synucleinopathies. Various aSyn conformations and post-translationally modified forms accumulate in pathological inclusions and vary in abundance among these disorders. Relying on antibodies that have not been assessed for their ability to detect the diverse forms of aSyn may lead to inaccurate estimations of aSyn pathology in human brains or disease models. To address this challenge, we developed and characterized an expanded antibody panel that targets different sequences and post-translational modifications along the length of aSyn, and that recognizes all monomeric, oligomeric, and fibrillar aSyn conformations. Next, we profiled aSyn pathology across sporadic and familial Lewy body diseases (LBDs) and reveal heterogeneous forms of aSyn pathology, rich in Serine 129 phosphorylation, Tyrosine 39 nitration and N- and C-terminal tyrosine phosphorylations, scattered both to neurons and glia. In addition, we show that aSyn can become hyperphosphorylated during processes of aggregation and inclusion maturation in neuronal and animal models of aSyn seeding and spreading. The validation pipeline we describe for these antibodies paves the way for systematic investigations into aSyn pathological diversity in the human brain, peripheral tissues, as well as in cellular and animal models of synucleinopathies.

Neuronal growth on high-aspect-ratio diamond nanopillar arrays for biosensing applications.

Monitoring neuronal activity with simultaneously high spatial and temporal resolution in living cell cultures is crucial to advance understanding of the development and functioning of our brain, and to gain further insights in the origin of brain disorders. While it has been demonstrated that the quantum sensing capabilities of nitrogen-vacancy (NV) centers in diamond allow real time detection of action potentials from large neurons in marine invertebrates, quantum monitoring of mammalian neurons (presenting much smaller dimensions and thus producing much lower signal and requiring higher spatial resolution) has hitherto remained elusive. In this context, diamond nanostructuring can offer the opportunity to boost the diamond platform sensitivity to the required level. However, a comprehensive analysis of the impact of a nanostructured diamond surface on the neuronal viability and growth was lacking. Here, we pattern a single crystal diamond surface with large-scale nanopillar arrays and we successfully demonstrate growth of a network of living and functional primary mouse hippocampal neurons on it. Our study on geometrical parameters reveals preferential growth along the nanopillar grid axes with excellent physical contact between cell membrane and nanopillar apex. Our results suggest that neuron growth can be tailored on diamond nanopillars to realize a nanophotonic quantum sensing platform for wide-field and label-free neuronal activity recording with sub-cellular resolution.

Electrochemical sensors for the detection of SARS-CoV-2 virus.

Coronavirus (COVID-19), a deadly pandemic has spread worldwide and created many global health issues. Though methods of its detection are being continuously developed for the early detection and monitoring of COVID-19, still there is need for more novel methods. The presently used methods include rapid antigen tests, serological surveys, reverse transcription-polymerase chain reaction (RT-PCR), artificial intelligence-based techniques, and assays based on sensors/biosensors. Of all these, RT-PCR test has high sensitivity and specificity though it requires more time for testing and need for skilled technicians. Recently, electrochemical sensors have been developed for rapid monitoring and detection of SARS-CoV-2 from the patient's biological fluid samples. This review covers the recently developed electrochemical sensors that are focused on the detection of viral nucleic acid, immunoglobulin, antigen, and the entire viral particles. In addition, we also compare and assess their detection limits, sensitivities and specificities for the identification and monitoring of COVID-19. Furthermore, this review will address the best practices for the development of electrochemical sensors such as electrode fouling, limit of detection/limit of quantification determination and verification.

Reverse engineering Lewy bodies: how far have we come and how far can we go?

Lewy bodies (LBs) are α-synuclein (α-syn)-rich intracellular inclusions that are an important pathological hallmark of Parkinson disease and several other neurodegenerative diseases. Increasing evidence suggests that the aggregation of α-syn has a central role in LB formation and is one of the key processes that drive neurodegeneration and pathology progression in Parkinson disease. However, little is known about the mechanisms underlying the formation of LBs, their biochemical composition and ultrastructural properties, how they evolve and spread with disease progression, and their role in neurodegeneration. In this Review, we discuss current knowledge of α-syn pathology, including the biochemical, structural and morphological features of LBs observed in different brain regions. We also review the most used cellular and animal models of α-syn aggregation and pathology spreading in relation to the extent to which they reproduce key features of authentic LBs. Finally, we provide important insights into molecular and cellular determinants of LB formation and spreading, and highlight the critical need for more detailed and systematic characterization of α-syn pathology, at both the biochemical and structural levels. This would advance our understanding of Parkinson disease and other neurodegenerative diseases and allow the development of more-reliable disease models and novel effective therapeutic strategies.

How specific are the conformation-specific α-synuclein antibodies? Characterization and validation of 16 α-synuclein conformation-specific antibodies using well-characterized preparations of α-synuclein monomers, fibrils and oligomers with distinct structures and morphology.

Increasing evidence suggests that alpha-synuclein (α-syn) oligomers are obligate intermediates in the pathway involved in α-syn fibrillization and Lewy body (LB) formation, and may also accumulate within LBs in Parkinson's disease (PD) and other synucleinopathies. Therefore, the development of tools and methods to detect and quantify α-syn oligomers has become increasingly crucial for mechanistic studies to understand their role in PD, and to develop new diagnostic methods and therapies for PD and other synucleinopathies. The majority of these tools and methods rely primarily on the use of aggregation state-specific or conformation-specific antibodies. Given the impact of the data and knowledge generated using these antibodies on shaping the foundation and directions of α-syn and PD research, it is crucial that these antibodies are thoroughly characterized, and their specificity or ability to capture diverse α-syn species is tested and validated. Herein, we describe an antibody characterization and validation pipeline that allows a systematic investigation of the specificity of α-syn antibodies using well-defined and well-characterized preparations of various α-syn species, including monomers, fibrils, and different oligomer preparations that are characterized by distinct morphological, chemical and secondary structure properties. This pipeline was used to characterize 18 α-syn antibodies, 16 of which have been reported as conformation- or oligomer-specific antibodies, using an array of techniques, including immunoblot analysis (slot blot and Western blot), a digital ELISA assay using single molecule array technology and surface plasmon resonance. Our results show that i) none of the antibodies tested are specific for one particular type of α-syn species, including monomers, oligomers or fibrils; ii) all antibodies that were reported to be oligomer-specific also recognized fibrillar α-syn; and iii) a few antibodies showed high specificity for oligomers and fibrils but did not bind to monomers. These findings suggest that the great majority of α-syn aggregate-specific antibodies do not differentiate between oligomers and fibrils, thus highlighting the importance of exercising caution when interpreting results obtained using these antibodies. Our results also underscore the critical importance of the characterization and validation of antibodies before their use in mechanistic studies and as diagnostic tools or therapeutic agents. This will not only improve the quality and reproducibility of research and reduce costs but will also reduce the number of therapeutic antibody failures in the clinic.

Inhibition of histone deacetylation rescues phenotype in a mouse model of Birk-Barel intellectual disability syndrome.

Mutations in the actively expressed, maternal allele of the imprinted KCNK9 gene cause Birk-Barel intellectual disability syndrome (BBIDS). Using a BBIDS mouse model, we identify here a partial rescue of the BBIDS-like behavioral and neuronal phenotypes mediated via residual expression from the paternal Kcnk9 (Kcnk9pat) allele. We further demonstrate that the second-generation HDAC inhibitor CI-994 induces enhanced expression from the paternally silenced Kcnk9 allele and leads to a full rescue of the behavioral phenotype suggesting CI-994 as a promising molecule for BBIDS therapy. Thus, these findings suggest a potential approach to improve cognitive dysfunction in a mouse model of an imprinting disorder.