Proteomics of Huntington's disease-affected human embryonic stem cells reveals an evolving pathology involving mitochondrial dysfunction and metabolic disturbances.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the Huntingtin gene, where excessive (≥ 36) CAG repeats encode for glutamine expansion in the huntingtin protein. Research using mouse models and human pathological material has indicated dysfunctions in a myriad of systems, including mitochondrial and ubiquitin/proteasome complexes, cytoskeletal transport, signaling, and transcriptional regulation. Here, we examined the earliest biochemical and pathways involved in HD pathology. We conducted a proteomics study combined with immunocytochemical analysis of undifferentiated HD-affected and unaffected human embryonic stem cells (hESC). Analysis of 1883 identifications derived from membrane and cytosolic enriched fractions revealed mitochondria as the primary dysfunctional organ in HD-affected pluripotent cells in the absence of significant differences in huntingtin protein. Furthermore, on the basis of analysis of 645 proteins found in neurodifferentiated hESC, we show a shift to transcriptional dysregulation and cytoskeletal abnormalities as the primary pathologies in HD-affected cells differentiating along neural lineages in vitro. We also show this is concomitant with an up-regulation in expression of huntingtin protein in HD-affected cells. This study demonstrates the utility of a model that recapitulates HD pathology and offers insights into disease initiation, etiology, progression, and potential therapeutic intervention.

Glutamate transporter variants reduce glutamate uptake in Alzheimer's disease.

A characteristic of Alzheimer's disease (AD) is that neuron populations in the temporal, frontal, and parietal cortices are selectively vulnerable. Several neurotransmitters have been proposed to play roles in neural destruction as AD progresses, including glutamate. Failure to clear the synaptic cleft of glutamate can overstimulate postsynaptic glutamate receptors, promoting neuronal death. Excitatory amino acid transporter 2 (EAAT2), which is concentrated in perisynaptic astrocytes, performs 90% of glutamate uptake in mammalian central nervous system. Alternative splicing of EAAT2 mRNA could regulate glutamate transport in normal and disease states. We report disease- and pathology-specific variations in EAAT2 splice variant expression in AD brain obtained at autopsy. While wild type EAAT2 showed a global reduction in expression, brain regions susceptible to neuronal loss demonstrated greater expression of transcripts that reduced glutamate transport in an in vitro assay. Functional splice variant EAAT2b showed no significant variation with disease state. These results have implications for the treatment of AD as modulators of EAAT2 splicing and/or glutamate uptake would augment current therapies aimed at blocking glutamate receptors.

Derivation of Huntington's disease-affected human embryonic stem cell lines.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an expansion of cytosine-adenine-guanine (CAG) repeats in the Huntingtin gene Htt. To facilitate research into HD, we have derived 4 human embryonic stem cell (hESC) lines containing ≥ 40 CAG repeats in exon 1 of Htt: SIVF017-HD (CAG40), SIVF018-HD (CAG46), SIVF020-HD (CAG48), and SIVF046-HD (CAG45). Additionally, we have derived a normal sibling-matched control for SIVF020-HD, cell line SIVF019. All 5 hESC lines had a normal karyotype, expressed pluripotency markers including Oct4, SSEA3, and Tra-1-81, and could be maintained in culture for multiple (>40) passages. Teratoma studies revealed that the hESC lines were capable of differentiating into cells representative of the 3 germ layers. Furthermore, in vitro neuronal differentiation experiments have confirmed that the hESC lines were able to generate MAP2-positive neuronal cells that express the Htt protein. Combined, these experiments confirm that the cell lines represent pluripotent stem cell lines. These HD-affected hESC lines will be made available to biomedical research laboratories and will provide a valuable tool to investigate the mechanisms and potential treatments for HD.

Housekeepers for accurate transcript expression analysis in Alzheimer's disease autopsy brain tissue.

BACKGROUND: Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a popular technique for mRNA expression studies. Normalization to an endogenous reference transcript (housekeeper) is widely used to correct for differences in loading and RNA quality. Alzheimer's disease (AD) alters brain metabolism. The stability of housekeeper transcript expression must be carefully validated. METHODS: qRT-PCR was used to assess eight putative housekeeper transcripts in four brain regions from 15 control, 12 AD, and 10 AD/Lewy body disease (LBD) cases. RESULTS: RNA quality is lower in AD and AD/LBD than in controls. Frequently used housekeepers such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-actin had lower overall expression in AD and AD/LBD cases than in controls. RPL13 and 18S were the most stably expressed housekeepers tested. Synaptophysin and glial fibrillary acidic protein were used to evaluate normalized quantification. By using different housekeepers we confirmed that synaptophysin expression was down-regulated in AD cases, whereas glial fibrillary acidic protein expression was increased. CONCLUSIONS: Among all candidates tested, RPL13 was the best housekeeper for qRT-PCR studies in autopsy brain tissue samples from controls and AD cases. RNA quality should be assessed and data normalized on this index as well.