Fen-Biao Gao

ESCRT-III Dysfunction Causes Autophagosome Accumulation and Neurodegeneration

Defects in the endosomal-lysosomal pathway have been implicated in a number of neurodegenerative disorders. A key step in the endocytic regulation of transmembrane proteins occurs in a subset of late-endosomal compartments known as multivesicular bodies (MVBs), whose formation is controlled by endosomal sorting complex required for transport (ESCRT). The roles of ESCRT in dendritic maintenance and neurodegeneration remain unknown. Here, we show that mSnf7-2, a key component of ESCRT-III, is highly expressed in most mammalian neurons. Loss of mSnf7-2 in mature cortical neurons caused retraction of dendrites and neuronal cell loss. mSnf7-2 binds to CHMP2B, another ESCRT-III subunit, in which a rare dominant mutation is associated with frontotemporal dementia linked to chromosome 3 (FTD3). Ectopic expression of the mutant protein CHMP2B(Intron5) also caused dendritic retraction prior to neurodegeneration. CHMP2B(Intron5) was associated more avidly than CHMP2B(WT) with mSnf7-2, resulting in sequestration of mSnf7-2 in ubiquitin-positive late-endosomal vesicles in cortical neurons. Moreover, loss of mSnf7-2 or CHMP2B(Intron5) expression caused the accumulation of autophagosomes in cortical neurons and flies. These findings indicate that ESCRT-III dysfunction is associated with the autophagy pathway, suggesting a novel neurodegeneration mechanism that may have important implications for understanding FTD and other age-dependent neurodegenerative diseases.

Accumulation of the cyclin‐dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation

Many types of vertebrate precursor cells divide a limited number of times before they stop and terminally differentiate. In no case is it known what causes them to stop dividing. We have been studying this problem in the proliferating precursor cells that give rise to post‐mitotic oligodendrocytes, the cells that make myelin in the central nervous system. We show here that two components of the cell cycle control system, cyclin D1 and the Cdc2 kinase, are present in the proliferating precursor cells but not in differentiated oligodendrocytes, suggesting that the control system is dismantled in the oligodendrocytes. More importantly, we show that the cyclin‐dependent kinase (Cdk) inhibitor p27 progressively accumulates in the precursor cells as they proliferate and is present at high levels in oligodendrocytes. Our findings are consistent with the possibility that the accumulation of p27 is part of both the intrinsic counting mechanism that determines when precursor cell proliferation stops and differentiation begins and the effector mechanism that arrests the cell cycle when the counting mechanism indicates it is time. The recent findings of others that p27‐deficient mice have an increased number of cells in all of the organs examined suggest that this function of p27 is not restricted to the oligodendrocyte cell lineage.

GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport.

The GGGGCC (G4C2) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia. The basis for pathogenesis is unknown. To elucidate the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2-repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. We show that these transgenic animals display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.

MicroRNA-9 Coordinates Proliferation and Migration of Human Embryonic Stem Cell-Derived Neural Progenitors

Human pluripotent stem cells offer promise for use in cell-based therapies for brain injury and diseases. However, their cellular behavior is poorly understood. Here we show that the expression of the brain-specific microRNA-9 (miR-9) is turned on in human neural progenitor cells (hNPCs) derived from human embryonic stem cells. Loss of miR-9 suppressed proliferation but promoted migration of hNPCs cultured in vitro. hNPCs without miR-9 activity also showed enhanced migration when transplanted into mouse embryonic brains or adult brains of a mouse model of stroke. These effects were not due to precocious differentiation of hNPCs. One of the key targets directly regulated by miR-9 encodes stathmin, which increases microtubule instability and whose expression in hNPCs correlates inversely with that of miR-9. Partial inhibition of stathmin activity suppressed the effects of miR-9 loss on proliferation and migration of human or embryonic rat neural progenitors. These results identify miR-9 as a novel regulator that coordinates the proliferation and migration of hNPCs.

Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had >1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization, two iPSC lines from each subject were selected, differentiated into postmitotic neurons, and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs, iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover, repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism.

Control of dendritic development by the Drosophila fragile X-related gene involves the small GTPase Rac1

Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 gene. How these mutations affect neuronal development and function remains largely elusive. We generated specific point mutations or small deletions in the Drosophila fragile X-related (Fmr1) gene and examined the roles of Fmr1 in dendritic development of dendritic arborization (DA) neurons in Drosophila larvae. We found that Fmr1 could be detected in the cell bodies and proximal dendrites of DA neurons and that Fmr1 loss-of-function mutations increased the number of higher-order dendritic branches. Conversely, overexpression of Fmr1 in DA neurons dramatically decreased dendritic branching. In dissecting the mechanisms underlying Fmr1 function in dendrite development, we found that the mRNA encoding small GTPase Rac1 was present in the Fmr1-messenger ribonucleoprotein complexes in vivo. Mosaic analysis with a repressor cell marker (MARCM) and overexpression studies revealed that Rac1 has a cell-autonomous function in promoting dendritic branching of DA neurons. Furthermore, Fmr1 and Rac1 genetically interact with each other in controlling the formation of fine dendritic branches. These findings demonstrate that Fmr1 affects dendritic development and that Rac1 is partially responsible for mediating this effect.

Control of Dendritic Field Formation in Drosophila: The Roles of Flamingo and Competition between Homologous Neurons

Neurons elaborate dendrites with stereotypic branching patterns, thereby defining their receptive fields. These branching patterns may arise from properties intrinsic to the neurons or competition between neighboring neurons. Genetic and laser ablation studies reported here reveal that different multiple dendritic neurons in the same dorsal cluster in the Drosophila embryonic PNS do not compete with one another for dendritic fields. In contrast, when dendrites from homologous neurons in the two hemisegments meet at the dorsal midline in larval stages, they appear to repel each other. The formation of normal dendritic fields and the competition between dendrites of homologous neurons require the proper expression level of Flamingo, a G protein-coupled receptor-like protein, in embryonic neurons. Whereas Flamingo functions downstream of Frizzled in specifying planar polarity, Flamingo-dependent dendritic outgrowth is independent of Frizzled.

Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity

The midbody is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. Midbodies persist in cells long after division as midbody derivatives (MBds), but their fate is unclear. Here we show that MBds are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells, induced pluripotent stem cells and potential cancer ‘stem cells’ in vivo and in vitro. MBd loss accompanies stem-cell differentiation, and involves autophagic degradation mediated by binding of the autophagic receptor NBR1 to the midbody protein CEP55. Differentiating cells and normal dividing cells do not accumulate MBds and possess high autophagic activity. Stem cells and cancer cells accumulate MBds by evading autophagosome encapsulation and exhibit low autophagic activity. MBd enrichment enhances reprogramming to induced pluripotent stem cells and increases the in vitro tumorigenicity of cancer cells. These results indicate unexpected roles for MBds in stem cells and cancer ‘stem cells’.

Context-dependent functions of specific microRNAs in neuronal development

MicroRNAs (miRNAs) are small noncoding RNAs that regulate multiple developmental processes at the post-transcriptional level. Recent rapid progresses have demonstrated critical roles for a number of miRNAs in neuronal development and function. In particular, miR-9 and miR-124 are specifically expressed in the mammalian nervous system, and their respective nucleotide sequences are 100% identical among many species. Yet, their expression patterns and mRNA targets are less conserved throughout evolution. As a consequence, these miRNAs exhibit diverse context-dependent functions in different aspects of neuronal development, ranging from early neurogenesis and neuronal differentiation to dendritic morphogenesis and synaptic plasticity. Some other neuronal miRNAs also exhibit context-dependent functions in development. Thus, post-transcriptional regulation of spatial and temporal expression levels of protein-coding genes by miRNAs contributes uniquely to the proper development and evolution of the complex nervous system.

Posttranscriptional control of neuronal development by microRNA networks

The proper development of the nervous system requires precise spatial and temporal control of gene expression at both the transcriptional and translational levels. In different experimental model systems, microRNAs (miRNAs) - a class of small, endogenous, noncoding RNAs that control the translation and stability of many mRNAs - are emerging as important regulators of various aspects of neuronal development. Further dissection of the in vivo physiological functions of individual miRNAs promises to offer novel mechanistic insights into the gene regulatory networks that ensure the precise assembly of a functional nervous system.