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

ABSTRACTAggregates 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.

Therapeutic targeting of autophagy in neurodegenerative and infectious diseases

Autophagy is a conserved process that uses double-membrane vesicles to deliver cytoplasmic contents to lysosomes for degradation. Although autophagy may impact many facets of human biology and disease, in this review we focus on the ability of autophagy to protect against certain neurodegenerative and infectious diseases. Autophagy enhances the clearance of toxic, cytoplasmic, aggregate-prone proteins and infectious agents. The beneficial roles of autophagy can now be extended to supporting cell survival and regulating inflammation. Autophagic control of inflammation is one area where autophagy may have similar benefits for both infectious and neurodegenerative diseases beyond direct removal of the pathogenic agents. Preclinical data supporting the potential therapeutic utility of autophagy modulation in such conditions is accumulating.

A comparison of the death induced by fungal invasion or toxic chemicals in cowpea epidermal cells. II. Responses induced by Erysiphe cichoracearum

Cowpea leaves were inoculated with the plantain powdery mildew fungus, Erysiphe cichoracearum, and fresh epidermal cells overlying veins were examined by light microscopy before being cleared or prepared for electron microscopy. Fungal appressoria usually formed a haustorium in the underlying nonhost cell, but only after what appeared to be an unsuccessful penetration attempt that induced a transient cytoplasmic aggregate, a ring of autofluorescence in the plant wall (best seen in cleared tissue), and in two examples observed ultrastructurally, a small penetration peg embedded in a callose-like papilla. The haustorium developed from a different penetration peg and elicited the death of the invaded cell. As reported for the death of cowpea epidermal cells elicited by CuCl2, cytoplasmic changes that occurred rapidly in fresh tissue after cytoplasmic streaming had stopped correlated closely with changes in ultrastructure. Compared with the CuCl2 study, microtubules and Golgi bodies disappeared faster and membranes appeared more disorganized. These data suggest that in cowpea epidermal cells, ultrastructural changes accurately predict the onset of cell death and may also reflect differences in its modes of induction.

Induced susceptibility and enhanced resistance at the cellular level in barley coleoptiles. II. Timing and localization of induced susceptibility in a single coleoptile cell and its transfer to an adjacent cell

The primary growth of both Erysiphe graminis de Candolle and E. pisi de Candolle on the same cell or adjacent cells of barley coleoptiles was observed by light microscopy to determine factors conditioning host cells toward susceptibility. Erysiphe pisi never successfully penetrated (0% efficiency) when it was the sole inoculum. By contrast it penetrated with varied efficiencies when initiation of cytoplasmic aggregates induced by it followed that of E. graminis by 4.0–19.0 h on the same coleoptile cells. When E. graminis induced a cytoplasmic aggregate 4.0–9.0 h earlier than E. pisi in a single coleoptile cell, induced susceptibility was minimal around the E. graminis penetration site, within 100 μm of the site, in the cell. When the time intervals were 9.25 – 14.0 h, induced susceptibility was maximal around the E. graminis penetration site, within 100 μm, and minimal when the fungi were more than 200 μm apart. When the time intervals were more than 14.0 h, induced susceptibility was evenly dispersed throughout the entire cell and transfer of susceptibility to laterally adjacent cells occurred only under these time conditions. Erysiphe pisi penetrations on coleoptile cells were influenced by its growth or the host cell's response before actual penetration occurred.