A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5' ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.

Amyotrophic lateral sclerosis (ALS) involves motor neuron degeneration, skeletal muscle atrophy, paralysis, and death. Mutations in Cu,Zn superoxide dismutase (SOD1) are one cause of the disease. Mice transgenic for mutated SOD1 develop symptoms and pathology similar to those in human ALS. To understand the disease mechanism, we developed a simple behavioral assay for disease progression in mice. Using this assay, we defined four stages of the disease in mice expressing G93A mutant SOD1. By studying mice with defined disease stages, we tied several pathological features into a coherent sequence of events leading to motor neuron death. We show that onset of the disease involves a sharp decline of muscle strength and a transient explosive increase in vacuoles derived from degenerating mitochondria, but little motor neuron death. Most motor neurons do not die until the terminal stage, approximately 9 weeks after disease onset. These results indicate that mutant SOD1 toxicity is mediated by damage to mitochondria in motor neurons, and this damage triggers the functional decline of motor neurons and the clinical onset of ALS. The absence of massive motor neuron death at the early stages of the disease indicates that the majority of motor neurons could be rescued after clinical diagnosis.

No abstract

Glial reaction is a common feature of neurodegenerative diseases. Recent studies have suggested that reactive astrocytes gain neurotoxic properties, but exactly how reactive astrocytes contribute to neurotoxicity remains to be determined. Here, we identify lipocalin 2 (lcn2) as an inducible factor that is secreted by reactive astrocytes and that is selectively toxic to neurons. We show that lcn2 is induced in reactive astrocytes in transgenic rats with neuronal expression of mutant human TAR DNA-binding protein 43 (TDP-43) or RNA-binding protein fused in sarcoma (FUS). Therefore, lcn2 is induced in activated astrocytes in response to neurodegeneration, but its induction is independent of TDP-43 or FUS expression in astrocytes. We found that synthetic lcn2 is cytotoxic to primary neurons in a dose-dependent manner, but is innocuous to astrocytes, microglia, and oligodendrocytes. Lcn2 toxicity is increased in neurons that express a disease gene, such as mutant FUS or TDP-43. Conditioned medium from rat brain slice cultures with neuronal expression of mutant TDP-43 contains abundant lcn2 and is toxic to primary neurons as well as neurons in cultured brain slice from WT rats. Partial depletion of lcn2 by immunoprecipitation reduced conditioned medium-mediated neurotoxicity. Our data indicate that reactive astrocytes secrete lcn2, which is a potent neurotoxic mediator.amyotrophic lateral sclerosis | astrocytosis G lial reaction is a common feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), Huntington disease, Parkinson disease, and Alzheimer's disease. Astrocytes and microglia become reactive during neurodegenerative processes (1, 2), and activated astrocytes may exhibit differential expression of astrocytic receptors, transporters, and transmitters; metabolic changes; and altered synthesis and release of proteins, chemokines, and cytokines (3-6). Controlled activation of astrocytes is considered beneficial to neurons (7), but overactive astrocytes can be harmful (8). Astrocytosis in neurodegeneration has been intensively studied, but exactly how reactive astrocytes contribute to neurotoxicity remains to be determined.Reactive astrocytes may lose neuroprotective functions or gain neurotoxic properties in neurodegenerative diseases. Astrocytes are responsible for the reuptake of the neurotransmitter glutamate, which is accomplished by excitatory amino acid transporter 2 (EAAT2 or GLT1) (9). In mice, GLT1 deficiency leads to synaptic glutamate accumulation and subsequent excitotoxicity (9). Astrocytes become reactive during neurodegeneration and gradually lose GLT1 function and expression (10-12). Stimulating GLT1 expression with antibiotics protects motor neurons in an ALS model (13). Because reactive astrocytes may lose their neuroprotective abilities, as observed during GLT1 deficiency (10-12), supplementing normal astrocytes to the areas of active neuropathology is expected to have a therapeutic effect. Indeed, transgenic rats with ALS are...