Visualisation of macropinosome maturation by the recruitment of sorting nexins

We report that phosphoinositol-binding sorting nexin 5 ( SNX5) associates with newly formed macropinosomes induced by EGF stimulation. We used the recruitment of GFP-SNX5 to macropinosomes to track their maturation. Initially, GFP-SNX5 is sequestered to discrete subdomains of the macropinosome; these subdomains are subsequently incorporated into highly dynamic, often branched, tubular structures. Time-lapse videomicroscopy revealed the highly dynamic extension of SNX5-labelled tubules and their departure from the macropinosome body to follow predefined paths towards the perinuclear region of the cell, before fusing with early endosomal acceptor membranes. The extension and departure of these tubular structures occurs rapidly over 5-10 minutes and is dependent upon intact microtubules. As the tubular structures depart from the macropinosome there is a reduction in the surface area and an increase in tension of the limiting membrane of the macropinosome. In addition to the recruitment of SNX5 to the macropinosome, Rab5, SNX1 and EEA1 are also recruited by newly formed macropinosomes, followed by the accumulation of Rab7. SNX5 forms heterodimers with SNX1 and this interaction is required for endosome association of SNX5. We propose that the departure of SNX5-positive tubules represents a rapid mechanism of recycling components from macropinosomes thereby promoting their maturation into Rab7-positive structures. Collectively these findings provide a detailed real-time characterisation of the maturation process of the macropinocytic endosome.

Clustering and spatial correlations of the neuronal cytoplasmic inclusions, astrocytic plaques and ballooned neurons in corticobasal degeneration

This study tested three hypotheses: (1) that there is clustering of the neuronal cytoplasmic inclusions (NCI), astrocytic plaques (AP) and ballooned neurons (BN) in corticobasal degeneration (CBD), (2) that the clusters of NCI and BN are not spatially correlated, and (3) that the lesions are correlated with disease ‘stage’. In 50% of the regions, clusters of lesions were 400–800 µm in diameter and regularly distributed parallel to the tissue boundary. Clusters of NCI and BN were larger in laminae II/III and V/VI, respectively. In a third of regions, the clusters of BN and NCI were negatively spatially correlated. Cluster size of the BN in the parahippocampal gyrus (PHG) was positively correlated with disease ‘stage’. The data suggest the following: (1) degeneration of the cortico-cortical pathways in CBD, (2) clusters of NCI and BN may affect different anatomical pathways and (3) BN may develop after the NCI in the PHG.

Spatial patterns of asynuclein positive glial cytoplasmic inclusions in multiple system atrophy

In cases of multiple system atrophy (MSA), glial cytoplasmic inclusions (GCI) were distributed randomly or present in large diffuse clusters (>1,600 μm in diameter) in most areas studied. These spatial patterns contrast with those reported for filamentous neuronal inclusions in the tauopathies and α-synucleinopathies. © 2003 Movement Disorder Society.

The spatial patterns of the tau immunopositive neuronal cytoplasmic inclusions (NCI) in the tauopathies

Tau positive neuronal cytoplasmic inclusions (NCI) are the ‘hallmark’ pathological feature of several neurodegenerative diseases collectively known as the tauopathies. This study compared the spatial patterns of various types of NCI in selected tauopathies including the neurofibrillary tangles (NFT) in Alzheimer's disease (AD) and progressive supranuclear palsy (PSP), Pick bodies (PB) in Pick’s disease (PiD), and the tau positive (tau+) neurons in corticobasal degeneration (CBD). In the cerebral cortex of these disorders, the tau+ NCI were distributed in clusters and in a significant proportion of analyses, the clusters were distributed with a regular periodicity parallel to the pia mater. The inclusions in AD, PiD and CBD exhibited a similar range of spatial patterns but in PSP were less frequently clustered and more frequently randomly distributed. In gyri where the NCI were clustered, there was a significant difference in mean cluster size between disorders. Hence, clusters of NFT in AD were larger than those in PSP and the tau+ neurons in CBD and clusters of PB in PiD were larger than the tau+ neurons in CBD and the NFT in PSP. The cluster size of the tau+ neurons in CBD was similar to the NFT in PSP. The data suggest that the formation of clusters of NCI, regularly distributed parallel to the pia mater, is a common feature of the tauopathies indicating similar patterns of cortical degeneration and pathogenic mechanisms across different diseases. Furthermore, the data suggest that cortical degeneration affecting the short and long cortico-cortical pathways may be a characteristic of the tauopathies.

Transglutaminase 2 is involved in autophagosome maturation

Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but it is enhanced in response to stress, e.g. nutrient deprivation, hypoxia, mitochondrial dysfunction and infection. "Tissue" transglutaminase (TG2) accumulates, both in vivo and in vitro, to high levels in cells under stressful conditions. Therefore, in this study, we investigated whether TG2 could also play a role in the autophagic process. To this end, we used TG2 knockout mice and cell lines in which the enzyme was either absent or overexpressed. The ablation of TG2 protein both in vivo and in vitro, resulted in an evident accumulation of microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) on pre-autophagic vesicles, suggesting a marked induction of autophagy. By contrast, the formation of the acidic vesicular organelles in the same cells was very limited, indicating an impairment of the final maturation of autophagolysosomes. In fact, the treatment of TG2 proficient cells with NH4Cl, to inhibit lysosomal activity, led to a marked accumulation of LC3 II and damaged mitochondria similar to what we observed in TG2-deficient cells. These data indicate a role for TG2-mediated post-translational modifications of proteins in the maturation of autophagosomes accompanied by the accumulation of many damaged mitochondria.

Spatial patterns of FUS-immunoreactive neuronal cytoplasmic inclusions (NCI) in neuronal intermediate filament inclusion disease (NIFID)

Neuronal intermediate filament inclusion disease (NIFID), a rare form of frontotemporal lobar degeneration (FTLD), is characterized neuropathologically by focal atrophy of the frontal and temporal lobes, neuronal loss, gliosis, and neuronal cytoplasmic inclusions (NCI) containing epitopes of ubiquitin and neuronal intermediate filament (IF) proteins. Recently, the 'fused in sarcoma' (FUS) protein (encoded by the FUS gene) has been shown to be a component of the inclusions of NIFID. To further characterize FUS proteinopathy in NIFID, we studied the spatial patterns of the FUS-immunoreactive NCI in frontal and temporal cortex of 10 cases. In the cerebral cortex, sectors CA1/2 of the hippocampus, and the dentate gyrus (DG), the FUS-immunoreactive NCI were frequently clustered and the clusters were regularly distributed parallel to the tissue boundary. In a proportion of cortical gyri, cluster size of the NCI approximated to those of the columns of cells was associated with the cortico-cortical projections. There were no significant differences in the frequency of different types of spatial patterns with disease duration or disease stage. Clusters of NCI in the upper and lower cortex were significantly larger using FUS compared with phosphorylated, neurofilament heavy polypeptide (NEFH) or a-internexin (INA) immunohistochemistry (IHC). We concluded: (1) FUS-immunoreactive NCI exhibit similar spatial patterns to analogous inclusions in the tauopathies and synucleinopathies, (2) clusters of FUS-immunoreactive NCI are larger than those revealed by NEFH or ???, and (3) the spatial patterns of the FUS-immunoreactive NCI suggest the degeneration of the cortico-cortical projections in NIFID.