The data we report in this study concern the types, location, numbers, forms, and composition of microscopic huntingtin aggregates in brain tissues from humans with different grades of Huntington's disease (HD). We have developed a fusion protein antibody against the first 256 amino acids that preferentially recognizes aggregated huntingtin and labels many more aggregates in neuronal nuclei, perikarya, and processes in human brain than have been described previously. Using this antibody and human brain tissue ranging from presymptomatic to grade 4, we have compared the numbers and locations of nuclear and neuropil aggregates with the known patterns of neuronal death in HD. We show that neuropil aggregates are much more common than nuclear aggregates and can be present in large numbers before the onset of clinical symptoms. There are also many more aggregates in cortex than in striatum, where they are actually uncommon. Although the striatum is the most affected region in HD, only 1-4% of striatal neurons in all grades of HD have nuclear aggregates. Neuropil aggregates, which we have identified by electron microscopy to occur in dendrites and dendritic spines, could play a role in the known dendritic pathology that occurs in HD. Aggregates increase in size in advanced grades, suggesting that they may persist in neurons that are more likely to survive. Ubiquitination is apparent in only a subset of aggregates, suggesting that ubiquitin-mediated proteolysis of aggregates may be late or variable.

Fragile X syndrome, a leading cause of inherited mental retardation, is attributable to the unstable expansion of a CGGrepeat within the FMR1 gene that results in the absence of the encoded protein. The fragile X mental retardation protein (FMRP) is a ribosome-associated RNA-binding protein of uncertain function that contains nuclear localization and export signals. We show here detailed cellular localization studies using both biochemical and immunocytochemical approaches. FMRP was highly expressed in neurons but not glia throughout the rat brain, as detected by light microscopy. Although certain structures, such as hippocampus, revealed a strong signal, the regional variation in staining intensity appeared to be related to neuron size and density. In human cell lines and mouse brain, FMRP co-fractionated primarily with polysomes and rough endoplasmic reticulum. Ultrastructural studies in rat brain revealed high levels of FMRP immunoreactivity in neuronal perikarya, where it is concentrated in regions rich in ribosomes, particularly near or between rough endoplasmic reticulum cisternae. Immunogold studies also provided evidence of nucleocytoplasmic shuttling of FMRP, which was localized in neuronal nucleoplasm and within nuclear pores. Moreover, labeling was observed in large-and small-caliber dendrites, in dendritic branch points, at the origins of spine necks, and in spine heads, all known locations of neuronal polysomes. Dendritic localization, which was confirmed by co-fractionation of FMRP with synaptosomal ribosomes, suggests a possible role of FMRP in the translation of proteins involved in dendritic structure or function and relevant for the mental retardation occurring in fragile X syndrome.

The precise localization of Dl and D2 dopamine receptors within striatal neurons and circuits is crucial information for further understanding dopamine pharmacology. We have used subtype specific polyclonal and monoclonal antibodies against Dl and D2 dopamine receptors to determine their cellular and subcellular distributions, their colocalization, and their differential connectivity with motor cortical afferents labeled either by lesion-induced degeneration or by anterograde transport of biotinylated dextrans. Dl and D2 are primarily expressed in medium-sized neurons and spiny dendrites. Axon terminals containing Dl were rare whereas DS-immunoreactive axon terminals forming symmetrical synapses with dendrites and spines were common. In 2 p.m sections, Dl was localized to 53% of neurons, and D2 to 48% of neurons, while mixing Dl and D2 antibodies labeled 78%. By electron microscopy, Dl was localized to 43% of dendrites and 38% of spines while D2 was localized to 38% of dendrites and 48% of spines. Combining Dl and D2 antibodies resulted in the labeling of 88.5% of dendrites and 92.6% of spines. Using different chromogens for Dl and D2, colocalization was not observed. lpsilateral motor corticostriatal afferents were primarily axospinous and significantly more synapsed with Dl than DS-positive spines (65% vs 47%). Contralateral motor corticostriatal afferents were frequently axodendritic and no difference in their frequency of synapses with Dl and D2 dendrites and spines was observed. These findings demonstrate differential patterns of expression of Dl and D2 receptorsin striatal neurons and axon terminals and their differential involvement in motor corticostriatal circuits.[

Huntington's disease (HD) is caused by an expanded N-terminal glutamine tract that endows huntingtin with a striatal-selective structural property ultimately toxic to medium spiny neurons. In precise genetic models of juvenile HD, HdhQ92 and HdhQ111 knock-in mice, long polyglutamine segments change huntingtin's physical properties, producing HD-like in vivo correlates in the striatum, including nuclear localization of a version of the full-length protein predominant in medium spiny neurons, and subsequent formation of N-terminal inclusions and insoluble aggregate. These changes show glutamine length dependence and dominant inheritance with recruitment of wild-type protein, critical features of the altered HD property that strongly implicate them in the HD disease process and that suggest alternative pathogenic scenarios: the effect of the glutamine tract may act by altering interaction with a critical cellular constituent or by depleting a form of huntingtin essential to medium spiny striatal neurons.

SUMMARY How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) is commonly upregulated in human cancers. GDH1 is important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate (α-KG) and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a ROS scavenging enzyme glutathione peroxidase 1 (GPx1). Targeting GDH1 by shRNA or a small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth.