Kon, T. | Search bioRxiv

bioRxiv2023

Neuronal SNCA transcription during Lewy body formation

BackgroundMisfolded -synuclein (-syn) is believed to contribute to neurodegeneration in Lewy body disease (LBD) based on considerable evidence including a gene-dosage effect observed in relation to point mutations and multiplication of SNCA in familial Parkinsons disease. A contradictory concept proposes early loss of the physiological -syn as the major driver of neurodegeneration. There is a paucity of data on SNCA transcripts in various -syn immunoreactive cytopathologies. MethodsSNCA transcripts in neurons without and with various -syn immunoreactive cytopathologies in the substantia nigra and amygdala in LBD (n = 5) were evaluated using RNAscope combined with immunofluorescence for disease-associated -syn. Single-nucleus RNA sequencing was performed to elucidate cell-type specific SNCA expression in non-diseased frontal cortex (n = 3). ResultsSNCA transcripts in neurons with punctate -syn immunoreactivity were preserved both in the substantia nigra and amygdala but were reduced in neurons with compact -syn inclusions. Only single SNCA transcripts were detected in astrocytes with or without -syn immunoreactivity in the amygdala. Single-nucleus RNA sequencing revealed that excitatory and inhibitory neurons, oligodendrocyte progenitor cells, oligodendrocytes, and homeostatic microglia expressed SNCA transcripts, while expression was largely absent in astrocytes and microglia. ConclusionsThe preserved cellular SNCA expression in the more abundant non-Lewy body type -syn cytopathologies provides a pool for local protein production that can aggregate and serve as a seed for misfolded -syn. Successful segregation of disease-associated -syn is associated with the exhaustion of SNCA production in the terminal cytopathology, the Lewy body. Our observations support a therapeutic strategy incorporating a finely tuned dual approach targeting the elimination of misfolded -syn along with the reduction of the SNCA transcription to avoid feeding of pathological -syn seeding.

pathology↗

bioRxiv2022

Key residue on cytoplasmic dynein for asymmetric unbinding and unidirectional movement along microtubule

Cytoplasmic dynein 1 is almost exclusively responsible for intracellular transport toward the minus-end of microtubules in animal cells. One of the key factors for the unidirectional movement of dynein is the asymmetry of the unbinding of the motor from the microtubule when an external load is applied; it dissociates more easily from microtubules with minus-end directed loading than with plus-end directed loading. To elucidate the molecular basis for this property, we performed molecular dynamics simulations to identify the key residues responsible for asymmetry, which were then examined experimentally. First, we reproduced asymmetry in the unbinding behavior of dynein using coarse-grained simulations. Then, data analysis together with mutational analysis in silico predicted the specific residues that may be responsible for the asymmetry in unbinding. To examine this prediction, we expressed and purified recombinant dynein with mutations in either of the identified key residues. Consistent with the simulations, one of the mutants did not exhibit asymmetry in the in vitro unbinding assay. Moreover, the mutant dynein was able to bind and move diffusely along a microtubule but was unable to restrict its movement to the minus-end direction. Our results demonstrate both experimentally and theoretically how the key residue on the microtubule-binding domain generates asymmetry in unbinding, which is a critical mechanism for the unidirectional movement of dynein along a microtubule track. Significance StatementCytoplasmic dynein moves to the minus end of microtubules. This unidirectional dynein motility provides the driving force for various cellular activities including vesicle transport, organelle positioning and cell division. One of the key factors for dynein to exhibit unidirectional movement is the asymmetry of unbinding of dynein from the microtubule depending on the direction of external load. By combining computational simulations and in vitro experiments, we identified a residue responsible for the asymmetry. A point mutation at the residue indeed abolished unidirectional motility, highlighting the importance of the asymmetric unbinding property in dyneins unidirectional movement.

biophysics↗