Biology subjects

Dhavale, D. D.

Publications and source records attributed to Dhavale, D. D..

8 recordsLinked to original sources

bioRxiv2026

Intrastriatal injection of amplified Lewy body dementia (LBD) fibrils in mice results in accumulation of fibrils with LBD structure and neurodegeneration

The defining neuropathologic feature of Lewy body dementia (LBD) and Parkinson's disease (PD) is alpha-synuclein (Asyn) fibril accumulation in Lewy bodies (LBs) and Lewy neurites (LNs). Recently, we developed a method to amplify Asyn fibrils derived from human post-mortem LBD brain tissue and determined that the structure of amplified Asyn fibrils is highly similar to the structure of fibrils directly isolated from LBD brain tissue. We examined whether injection of these fibrils into humanized Asyn mice recapitulates pathologic Asyn fibril accumulation with human LBD Asyn fibril structure. We utilized mice containing a null allele in the mouse Snca gene and a human SNCA transgene. Intrastriatal injection of amplified Asyn fibrils derived from 3 different LBD autopsy cases resulted in cytoplasmic and neuritic phospho-Asyn accumulation in the striatum, cortex, and amygdala bilaterally at 3mpi. Fibril injections produced a reduction in the number of dopaminergic neurons in the substantia nigra, with LB-like inclusions in surviving neurons, and produced impaired sensorimotor function as measured on the accelerating rotarod test. Fibril accumulation increased 3.6-fold between 1 and 3 months post-injection (mpi) and was relatively stable between 3 and 5 mpi. We amplified fibrils from mouse brain tissue 5 mpi and collected single-particle cryo-EM data. High resolution 3D structural information could not be obtained because of the lack of helical twist, but comparison of the cryo-EM 2D class averages with those derived from amplified fibrils prepared from human LBD tissue revealed multiple matching classes, indicating that the Lewy fold is maintained in the fibrils that accumulate in mouse brain tissue. Our findings demonstrate an approach to model the accumulation of Asyn fibrils with LBD structure in mice, providing a platform for investigating disease mechanisms, imaging biomarkers, and therapeutics targeting Asyn in PD/LBD.

neuroscience↗

bioRxiv2026

In vitro-prepared A30P alpha-synuclein fibrils adopt the conserved and disease-relevant Greek key fold

The pathological hallmark of Parkinson Disease (PD) is the formation of the protein alpha-synuclein (Asyn) into {beta}-sheet rich, self-templating fibrils in the brain. Since the first atomic structure of wild-type Asyn fibrils was determined nearly a decade ago, several other in vitro structures of hereditary mutant fibrils and structures derived from post-mortem diseased patient tissue have been determined by solid-state nuclear magnetic resonance (SSNMR) spectroscopy and cryo-electron microscopy. These structures have not only expanded the library of structures available for computational modeling of drug binding and therapeutics development but have also given unprecedented insight into the disease specificity and structural polymorphism of Asyn fibrils. Here, we report the high-resolution SSNMR structure of the A30P hereditary mutant Asyn fibril, associated with early-onset PD. Our structural model is calculated using several thousand distance restraints derived from one sample, primarily sourced through 3D 13C-13C-13C correlation experiments. The structure adopts a Greek key topology yet does not include the P30 mutation site within the fibril core. We also introduce a comprehensive method for the rapid comparison of SSNMR spectra between Asyn polymorphs of known structure and validate the A30P fold. Lastly, we find that the structure is highly similar to many other experimental structures of both in vitro and ex vivo Asyn fibrils, including those with other hereditary point mutations, suggesting a conserved accessible fold.

biophysics↗

bioRxiv2025

Identification of Small Molecule Dimethyoxyphenyl Piperazine Inhibitors of Alpha-Synuclein Fibril Growth

Identification of Small Molecule Dimethyoxyphenol Piperazine Inhibitors of Alpha-Synuclein Fibril Growth Alpha-synuclein (asyn) fibril accumulation is the defining feature of Parkinson disease and is a target for disease-modifying treatments. One therapeutic strategy to reduce fibril accumulation is inhibition of asyn fibril growth. We developed a sensitive fluorescence-based fibril growth assay to screen for small molecule inhibitors. After validating the inhibition assay using a previously identified inhibitor, epigallocatechin-3-gallate, we identified compound 1 as a lead for inhibition of fibril growth. We analysed structure-activity relationships with analogs of 1 to optimize inhibition potency. Our results identified two dimethoxyphenyl piperazine analogs with more potent inhibition of in-vitro assembled fibrils. These analogs also inhibited the growth of asyn fibrils amplified from Lewy Body Disease brain tissue, further validating the inhibitor screening assay. Molecular docking studies indicate that these compounds can bind to the fibril ends, suggesting a potential capping mechanism through which these compounds inhibit the sequential association of monomeric asyn required for fibril growth.

neuroscience↗

bioRxiv2025

Alpha-Synuclein Fibril Structures Cluster into Distinct Classes

The accumulation of Alpha-synuclein (Asyn) fibrils is the defining pathologic feature in Parkinson Disease (PD), Lewy Body Dementia (LBD), and Multiple System Atrophy (MSA). As such, the process of Asyn fibril formation has been an important research area and fibrils themselves have become attractive targets for disease diagnosis and therapeutic intervention. Due to the presence of mixed populations of fibrillar proteins associated with neurodegenerative diseases in brain tissue, high-resolution structures of Asyn fibrils are essential for the design of high-specificity imaging and therapeutic agents. Approximately one hundred high-resolution solid-state NMR (SSNMR) spectroscopy and cryo-electron microscopy (cryo-EM) structures of Asyn fibrils have been deposited to the Protein Databank (PDB); intriguingly there is significant polymorphism among them. Understanding the molecular makeup and characteristic features of each structural polymorph can determine conserved structural motifs which can be used as templates to design ligands with high specificity for clinical use. Utilizing standard alignment tools and density-based clustering approaches, we objectively classify fibril structures by tertiary structure type. We find that 81% of the structures cluster into two polymorph classes. Within each class, additional subtle variations are observed which position sidechains in specific, conserved orientations, well poised as druggable targets. Furthermore, we find that the conserved structural motifs associated with each class are found in all but one published Asyn fibril structure. We consider these classifications and conserved motifs in the context of disease-relevant fibril structures and offer a perspective on the utility of in vitro fibrils as substrates for drug development and models for disease pathogenesis.

biophysics↗

bioRxiv2024

Seeding competent TDP-43 persists in human patient and mouse muscle

TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous system of some neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy (IBM). TDP-43 aggregates from ALS and FTD brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in IBM patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with IBM, IMNM and ALS we found that TDP-43 seeding capacity was specific to IBM. Surprisingly, TDP-43 seeding capacity anti-correlated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle disease. SummaryTDP-43 aggregate seeds persist in mouse and human skeletal muscle independent of large TDP-43 inclusions.

pathology↗

bioRxiv2023

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.

neuroscience↗

bioRxiv2023

13C and 15N Resonance Assignments of Alpha Synuclein Fibrils Amplified from Lewy Body Dementia Tissue

Fibrils of the protein -synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

biophysics↗

bioRxiv2018

TDP-43 oligomers detected as initial intermediate species during aggregate formation

Aggregates of the RNA binding protein TDP-43 are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are neurodegenerative disorders with overlapping clinical, genetic and pathological features. Mutations in the TDP-43 gene are causative of ALS, supporting its central role in pathogenesis. The process of TDP-43 aggregation remains poorly understood and whether this includes formation of intermediate complexes is unknown. We characterized aggregates derived from purified TDP-43 as a function of time and analyzed them under semi-denaturing conditions. Our assays identified oligomeric complexes at the initial time points prior to the formation of large aggregates, suggesting that ordered oligomerization is an intermediate step of TDP-43 aggregation. In addition, we analyzed liquid-liquid phase separation of TDP-43 and detected similar oligomeric assembly upon the maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, ALS-linked mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that the aggregates generated from purified protein seed intracellular aggregation, which is detected by established markers of TDP-43 pathology. Remarkably, cytoplasmic aggregate propagation is detected earlier with A315T and M337V and is 50% more widespread than with wild-type aggregates. Our findings provide evidence for a controlled process of TDP-43 self-assembly into intermediate structures that provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.

biochemistry↗