Non-amyloid, ubiquitinated cytoplasmic inclusions containing TDP-43 and its C-terminal fragments are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder, and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Importantly, TDP-43 mutations are linked to sporadic and non-SOD1 familial ALS. However, TDP-43 is not the only protein in disease-associated inclusions, and whether TDP-43 misfolds or is merely sequestered by other aggregated components is unclear. Here, we report that, in the absence of other components, TDP-43 spontaneously forms aggregates bearing remarkable ultrastructural similarities to TDP-43 deposits in degenerating neurons of ALS FTLD-U patients. The C-terminal domain of TDP-43 is critical for spontaneous aggregation. Several ALS-linked TDP-43 mutations within this domain (Q331K, M337V, Q343R, N345K, R361S, and N390D) increase the number of TDP-43 aggregates and promote toxicity in vivo. Importantly, mutations that promote toxicity in vivo accelerate aggregation of pure TDP-43 in vitro. Thus, TDP-43 is intrinsically aggregation-prone, and its propensity for toxic misfolding trajectories is accentuated by specific ALS-linked mutations.TDP-43 is a ubiquitously expressed and highly conserved metazoan nuclear protein (1), which contains two RNA recognition motifs (RRMs) 3 and a glycine-rich region in its C-terminal domain (see Fig. 1A). TDP-43 function is uncertain, but it likely plays important roles in pre-mRNA splicing and transcriptional repression (2, 3). In ALS and FTLD-U, TDP-43 is depleted from the nucleus and accumulates in ubiquitinated cytoplasmic inclusions (4). These and other situations of TDP-43 pathology, including some forms of Alzheimer and Parkinson diseases, are now known as TDP-43 proteinopathies (5). Importantly, mutations in the TDP-43 gene (TARDBP) are linked to sporadic and non-SOD1 familial ALS, implying that TDP-43 abnormalities are likely one cause of disease (6 -11). However, despite this synthesis of pathology and genetics, the mechanisms by which TDP-43 might contribute to disease remain unknown and controversial (12, 13).A key unresolved question is whether TDP-43 is inherently aggregation-prone or whether TDP-43 is sequestered by other aggregated components and is merely a marker of disease (13-16). Indeed, multiple proteins aside from TDP-43 are found in Sarkosyl-insoluble fractions from FTLD-U patients (14). Moreover, deconvolution imaging reveals that TDP-43 appears to be excluded from some regions of the ubiquitinated inclusions in ALS (15).Here, we assess TDP-43 aggregation in the absence of other components. We then define which domains of TDP-43 are important for this process and determine the direct effects of several ALS-linked TDP-43 mutations on TDP-43 misfolding and toxicity. Our findings bring to light several intrinsic properties of TDP-43 and ALS-linked TDP-43 mutants that likely play important roles in the aberrant TDP-43 proteostasis (17) that contributes to the pathogenesis of ALS,...

Summary L eucine R ich R epeat K inase 2 ( LRRK2 ) is the most commonly mutated gene in familial Parkinson’s disease (PD) 1 and is also linked to its idiopathic form 2 . LRRK2 is proposed to function in membrane trafficking 3 and co-localizes with microtubules 4 . Despite LRRK2’s fundamental importance for understanding and treating PD, there is limited structural information on it. Here we report the 3.5Å structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported 14Å cryo-electron tomography in situ structure 5 . We propose that the conformation of LRRK2’s kinase domain regulates its microtubule interaction, with a closed conformation favoring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin-1 and cytoplasmic dynein-1 in vitro . Kinase inhibitors that stabilize an open conformation relieve this interference and reduce LRRK2 filament formation in cells, while those that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

Alpha-synuclein is a small neuronal protein that is closely associated with the etiology of Parkinson's disease. Mutations in and alterations in expression levels of alpha-synuclein cause autosomal dominant early onset heredity forms of Parkinson's disease, and sporadic Parkinson's disease is defined in part by the presence of Lewy bodies and Lewy neurites that are composed primarily of alpha-synuclein deposited in an aggregated amyloid fibril state. The normal function of alpha-synuclein is poorly understood, and the precise mechanisms by which it leads to toxicity and cell death are also unclear. Although alpha-synuclein is a highly soluble, cytoplasmic protein, it binds to a variety of cellular membranes of different properties and compositions. These interactions are considered critical for at least some normal functions of alpha-synuclein, and may well play critical roles in both the aggregation of the protein and its mechanisms of toxicity. Here we review the known features of alpha-synuclein membrane interactions in the context of both the putative functions of the protein and of its pathological roles in disease.

SummaryThe protein-remodeling machine Hsp104 dissolves amorphous aggregates as well as ordered amyloid assemblies such as yeast prions. Force generation originates from a tandem AAA+ (ATPases associated with various cellular activities) cassette, but the mechanism and allostery of this action remain to be established. Our cryoelectron microscopy maps of Hsp104 hexamers reveal substantial domain movements upon ATP binding and hydrolysis in the first nucleotide-binding domain (NBD1). Fitting atomic models of Hsp104 domains to the EM density maps plus supporting biochemical measurements show how the domain movements displace sites bearing the substrate-binding tyrosine loops. This provides the structural basis for N- to C-terminal substrate threading through the central cavity, enabling a clockwise handover of substrate in the NBD1 ring and coordinated substrate binding between NBD1 and NBD2. Asymmetric reconstructions of Hsp104 in the presence of ATPγS or ATP support sequential rather than concerted ATP hydrolysis in the NBD1 ring.

Synapses continually replenish their synaptic vesicle (SV) pools while suppressing spontaneous fusion events, thus maintaining a high dynamic range in response to physiological stimuli. The presynaptic protein complexin can both promote and inhibit fusion through interactions between its alpha-helical domain and the SNARE complex. In addition, complexin’s C-terminal half is required for the inhibition of spontaneous fusion in worm, fly, and mouse, although the molecular mechanism remains unexplained. We show here that complexin’s C-terminal domain binds lipids through a novel protein motif, permitting complexin to inhibit spontaneous exocytosis in vivo by targeting complexin to SVs. We propose that the SV pool serves as a platform to sequester and position complexin where it can intercept the rapidly assembling SNAREs and control the rate of spontaneous fusion.