Significance: Parkinson’s disease (PD) is a neurodegenerative disease linked to the misfolding and aggregation of a protein called “α-synuclein.” α-Synuclein aggregates found in the brains of PD patients are called “Lewy bodies” and “Lewy neurites.” We discovered the existence of a type of α-synuclein aggregate, smaller than previously described and conformationally distinct, that we called “pα-syn*.” Pα-syn* was present in neuronal cultures and mice brains injected with recombinant α-synuclein fibrils as well as in the brains of PD patients. We showed that pα-syn* is made of trimmed α-synuclein resulting from a failed cellular attempt to degrade fibrillar α-synuclein aggregates. We found that pα-syn* is a major neurotoxic species inducing mitochondrial damage, fission, and mitophagy, therefore constituting a central player in PD pathogenesis.

Significance: In vivo fluorescence microscopy with resolution well beyond the diffraction limit entails complexities that challenge the attainment of sufficient image brightness and contrast. These challenges have so far hampered investigations of the nanoscale distributions of synaptic proteins in the living mouse. Here, we describe a combination of stimulated emission depletion microscopy and endogenous protein labeling, providing high-quality in vivo data of the key scaffolding protein PSD95 at the postsynaptic membrane, which frequently appeared in extended distributions rather than as isolated nanoclusters. Operating in the far-red to near-IR wavelength range, this combination promises reduced photostress compared with prior in vivo nanoscopy at much shorter wavelengths.

Ca²⁺ influx triggers the release of synaptic vesicles at the presynaptic active zone (AZ). A quantitative characterization of presynaptic Ca²⁺ signaling is critical for understanding synaptic transmission. However, this has remained challenging to establish at the required resolution. Here, we employ confocal and stimulated emission depletion (STED) microscopy to quantify the number (20–330) and arrangement (mostly linear 70 nm × 100–600 nm clusters) of Ca²⁺ channels at AZs of mouse cochlear inner hair cells (IHCs). Establishing STED Ca²⁺ imaging, we analyze presynaptic Ca²⁺ signals at the nanometer scale and find confined elongated Ca²⁺ domains at normal IHC AZs, whereas Ca²⁺ domains are spatially spread out at the AZs of bassoon-deficient IHCs. Performing 2D-STED fluorescence lifetime analysis, we arrive at estimates of the Ca²⁺ concentrations at stimulated IHC AZs of on average 25 µM. We propose that IHCs form bassoon-dependent presynaptic Ca²⁺-channel clusters of similar density but scalable length, thereby varying the number of Ca²⁺ channels amongst individual AZs.