Fragments of HdhQ150 mutant huntingtin form a soluble oligomer pool that declines with aggregate deposition upon aging.

Cleavage of the full-length mutant huntingtin (mhtt) protein into smaller, soluble aggregation-prone mhtt fragments appears to be a key process in the neuropathophysiology of Huntington's Disease (HD). Recent quantification studies using TR-FRET-based immunoassays showed decreasing levels of soluble mhtt correlating with an increased load of aggregated mhtt in the aging HdhQ150 mouse brain. To better characterize the nature of these changes at the level of native mhtt species, we developed a detection method that combines size exclusion chromatography (SEC) and time-resolved fluorescence resonance energy transfer (TR-FRET) that allowed us to resolve and define the formation, aggregation and temporal dynamics of native soluble mhtt species and insoluble aggregates in the brain of the HdhQ150 knock-in mouse.

A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein.

Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors.

Neurotrophin receptors TrkA and TrkC cause neuronal death whereas TrkB does not.

Neurons of the peripheral nervous system have long been known to require survival factors to prevent their death during development. But why they selectively become dependent on secretory molecules has remained a mystery, as is the observation that in the central nervous system, most neurons do not show this dependency. Using engineered embryonic stem cells, we show here that the neurotrophin receptors TrkA and TrkC (tropomyosin receptor kinase A and C, also known as Ntrk1 and Ntrk3, respectively) instruct developing neurons to die, both in vitro and in vivo.

Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs.

Adaptation to different levels of illumination is central to the function of the retina. Here, we demonstrate that levels of the miR-183/96/182 cluster, miR-204, and miR-211 are regulated by different light levels in the mouse retina. Concentrations of these microRNAs were downregulated during dark adaptation and upregulated in light-adapted retinas, with rapid decay and increased transcription being responsible for the respective changes.

Single-step detection of mutant huntingtin in animal and human tissues: a bioassay for Huntington's disease.

The genetic mutation causing Huntington's disease is a polyglutamine expansion in the huntingtin protein where more than 37 glutamines cause disease by formation of toxic intracellular fragments, aggregates, and cell death. Despite a clear pathogenic role for mutant huntingtin, understanding huntingtin expression during the presymptomatic phase of the disease or during disease progression has remained obscure. Central to clarifying the role in the pathomechanism of disease is the ability to easily and accurately measure mutant huntingtin in accessible human tissue samples as well as cell and animal models.

Embryonic stem cell-derived neurons as a cellular system to study gene function: lack of amyloid precursor proteins APP and APLP2 leads to defective synaptic transmission.

The in vitro generation of uniform populations of neurons from mouse embryonic stem cells (ESCs) provides a novel opportunity to study gene function in neurons. This is of particular interest when mutations lead to lethal in vivo phenotypes. Although the amyloid precursor protein (APP) and its proteolysis are regarded as key elements of the pathology of Alzheimer's disease, the physiological function of APP is not well understood and mice lacking App and the related gene Aplp2 die early postnatally without any obvious histopathological abnormalities.

Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors.

Cellular differentiation entails loss of pluripotency and gain of lineage- and cell-type-specific characteristics. Using a murine system that progresses from stem cells to lineage-committed progenitors to terminally differentiated neurons, we analyzed DNA methylation and Polycomb-mediated histone H3 methylation (H3K27me3). We show that several hundred promoters, including pluripotency and germline-specific genes, become DNA methylated in lineage-committed progenitor cells, suggesting that DNA methylation may already repress pluripotency in progenitor cells.

Sensitive biochemical aggregate detection reveals aggregation onset before symptom development in cellular and murine models of Huntington's disease.

A CAG-repeat gene expansion translated into a pathogenic polyglutamine stretch at the N-terminus of huntingtin triggers Huntington's Disease. Mutated huntingtin is predicted to adopt toxic properties mainly if aggregation-prone N-terminal fragments are released by proteolysis. Huntingtin-aggregates are indeed a major hallmark of this disorder and could represent useful markers of disease-onset or progression.

Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells.

A detailed protocol is described allowing the generation of essentially pure populations of glutamatergic neurons from mouse embryonic stem (ES) cells. It is based on the culture of ES cells that are kept undifferentiated by repeated splitting and subsequently amplified as non-adherent cell aggregates. Treatment with retinoic acid causes these ES cells to essentially become neural progenitors with the characteristics of Pax6-positive radial glial cells.

Identification of a lectin causing the degeneration of neuronal processes using engineered embryonic stem cells.

Unlike the mechanisms involved in the death of neuronal cell bodies, those causing the elimination of processes are not well understood owing to the lack of suitable experimental systems. As the neurotrophin receptor p75(NTR) is known to restrict the growth of neuronal processes, we engineered mouse embryonic stem (ES) cells to express an Ngfr (p75(NTR)) cDNA under the control of the Mapt locus (the gene encoding tau), which begins to be active when ES cell-derived progenitors start elongating processes. This caused a progressive, synchronous degeneration of all processes, and a prospective proteomic analysis showed increased levels of the sugar-binding protein galectin-1 in the p75(NTR)-engineered cells.

Developmental potential of defined neural progenitors derived from mouse embryonic stem cells.

The developmental potential of a uniform population of neural progenitors was tested by implanting them into chick embryos. These cells were generated from retinoic acid-treated mouse embryonic stem (ES) cells, and were used to replace a segment of the neural tube. At the time of implantation, the progenitors expressed markers defining them as Pax6-positive radial glial (RG) cells, which have recently been shown to generate most pyramidal neurons in the developing cerebral cortex.

Differentiation of mouse embryonic stem cells into a defined neuronal lineage.

Although it has long been known that cultured embryonic stem cells can generate neurons, the lineage relationships with their immediate precursors remain unclear. We report here that selection of highly proliferative stem cells followed by treatment with retinoic acid generated essentially pure precursors that markers identified as Pax-6-positive radial glial cells. As they do in vivo, these cells went on to generate neurons with remarkably uniform biochemical and electrophysiological characteristics.