Postmortem Huntington's Disease Research Articles

Gene expression changes in corticostriatal neurons of Huntington's Disease transgenic mice and postmortem human brains

Transcriptional dysregulation occurs in Huntington's Disease (HD). It affects striatal vulnerable neurons and neurons that are resistant against neurodegeneration (Zucker et al. 2005). The most affected brain region in HD besides the striatum is the cortex. Searching for causes that explain the selective neurodegeneration, we studied deep layer cortical neurons projecting to the striatum, which we isolated by Laser Capture Microdissection from sections of R6/2 transgenic mice and postmortem human HD brain. We have examined in detail both the mRNAs showing enrichment in the deep layer cortical cells and mRNAs showing most dramatic differential expression in the disease model. Cortical neurons from motor cortex (M1 and M2) of HD transgenic mice demonstrate significant changes in gene expression: 329 decreases and 94 increases in Affymetrix microarrays, 39000 mRNA probesets. Compared to cortical homogenate studies, several new mRNAs are detected in the microdissected deep layer cortical neurons. Comparing the detected murine changes to those in human postmortem HD brain by real-time PCR shows that some gene expression changes also occur in human HD tissue in the primary motor cortex (Brodmann Area 4) but not in the prefrontal association cortex (Brodmann Area 9). In deep layers of both R6/2 mice and postmortem HD human cortex, the expression of lin7b is significantly decreased. Lin7b is a structural polarity molecule implicated in protein sorting, intracellular transport, dendritic outgrowth and synaptic plasticity. We therefore postulate that corticostriatal neurons may be affected in HD by disturbance of polarity.

Contribution of nuclear and extranuclear polyQ to neurological phenotypes in mouse models of Huntington's disease

In postmortem Huntington's disease brains, mutant htt is present in both nuclear and cytoplasmic compartments. To dissect the impact of nuclear and extranuclear mutant htt on the initiation and progression of disease, we generated a series of transgenic mouse lines in which nuclear localization or nuclear export signal sequences have been placed N-terminal to the htt exon 1 protein carrying 144 glutamines. Our data indicate that the exon 1 mutant protein is present in the nucleus as part of an oligomeric or aggregation complex. Increasing the concentration of the mutant transprotein in the nucleus is sufficient for and dramatically accelerates the onset and progression of behavioral phenotypes. Furthermore, nuclear exon 1 mutant protein is sufficient to induce cytoplasmic neurodegeneration and transcriptional dysregulation. However, our data suggest that cytoplasmic mutant exon 1 htt, if present, contributes to disease progression.

Genome-wide expression profiling of human blood reveals biomarkers for Huntington's disease

Huntington's disease (HD) is an autosomal dominant disorder caused by an expansion of glutamine repeats in ubiquitously distributed huntingtin protein. Recent studies have shown that mutant huntingtin interferes with the function of widely expressed transcription factors, suggesting that gene expression may be altered in a variety of tissues in HD, including peripheral blood. Affymetrix and Amersham Biosciences oligonucleotide microarrays were used to analyze global gene expression in blood samples of HD patients and matched controls. We identified 322 mRNAs that showed significantly altered expression in HD blood samples, compared with controls (P < 0.0005), on two different microarray platforms. A subset of up-regulated mRNAs selected from this group was able to distinguish controls, presymptomatic individuals carrying the HD mutation, and symptomatic HD patients. In addition, early presymptomatic subjects showed gene expression profiles similar to those of controls, whereas late presymptomatic subjects showed altered expression that resembled that of symptomatic HD patients. These elevated mRNAs were significantly reduced in HD patients involved in a dose-finding study of the histone deacetylase inhibitor sodium phenylbutyrate. Furthermore, expression of the marker genes was significantly up-regulated in postmortem HD caudate, suggesting that alterations in blood mRNAs may reflect disease mechanisms observed in HD brain. In conclusion, we identified changes in blood mRNAs that clearly distinguish HD patients from controls. These alterations in mRNA expression correlate with disease progression and response to experimental treatment. Such markers may provide clues to the state of HD and may be of predictive value in clinical trials.

Prevention of cytosolic IAPs degradation: a potential pharmacological target in Huntington's Disease

Huntington's Disease (HD) is a neurodegenerative disorder caused by an abnormally expanded polyglutamine trait in the amino-terminal region of huntingtin. Pathogenic mechanisms involve a gained toxicity of mutant huntingtin and a potentially reduced neuroprotective function of the wild-type allele. Among the molecular abnormalities reported, HD cells are characterized by the presence of aggregates, transcriptional dysregulation, altered mitochondrial membrane potential and aberrant Ca++ handling. In addition, upon exposure to toxic stimuli, increased mitochondrial release of cytochrome C and activation of caspase-9 and caspase-3 are found in HD cells and tissue. Here we report that HTRA2 and Smac/DIABLO, two additional mitochondrial pro-apoptotic factors, are aberrantly released from brain-derived cells expressing mutant huntingtin. This event causes a reduction in levels of the cytosolic IAP1 (Inhibitor of Apoptosis Protein-1) and XIAP (X-linked inhibitor apoptosis) antiapoptotic IAP family members. Reduced IAP levels are also found in post-mortem HD brain tissue. Treatment with ucf101, a serine protease HTRA2 specific inhibitor, counteracts IAPs degradation in HD cells and increases their survival. These results point to the IAPs as potential pharmacological targets in Huntington's Disease.

Biochemical, Ultrastructural, and Reversibility Studies on Huntingtin Filaments Isolated from Mouse and Human Brain

Huntington's disease (HD) and eight additional inherited neurological disorders are caused by CAG triplet-repeat expansions leading to expanded polyglutamine-sequences in their respective proteins. These triplet-CAG repeat disorders have in common the formation of aberrant intraneuronal proteinaceous inclusions containing the expanded polyglutamine sequences. These aggregates have been postulated to contribute to pathogenesis caused by conformational toxicity, sequestration of other polyglutamine-containing proteins, or by interfering with certain enzymatic activities. Testing these hypotheses has been hampered by the difficulty to isolate these aggregates from brain. Here we report that polyglutamine aggregates can be isolated from the brain of the Tet/HD94 conditional mouse model of HD, by following a method based on high salt buffer homogenization, nonionic detergent extraction, and gradient fractionation. We then verified that the method can be successfully applied to postmortem HD brains. Immunoelectron microscopy, both in human and mouse samples, revealed that the stable component of the inclusions are mutant huntingtin-containing and ubiquitin-containing fibrils. Atomic-force microscopy revealed that these fibrils have a "beads on a string" morphology. Thus, they resemble the in vitro assembled filaments made of recombinant mutant-huntingtin, as well as the Abeta and alpha-synuclein amyloid protofibrils. Finally, by shutting down transgene expression in the Tet/HD94 conditional mouse model of HD, we were able to demonstrate that these filaments, although stable in vitro, are susceptible to revert in vivo, thus demonstrating that the previously reported reversal of ubiquitin-immunoreactive inclusions does not simply reflect disassembling of the inclusions into their constituent fibrils and suggesting that any associated conformational or protein-sequestration toxicity is also likely to revert.

Increased cell proliferation and neurogenesis in the adult human Huntington's disease brain.

Neurogenesis has recently been observed in the adult human brain, suggesting the possibility of endogenous neural repair. However, the augmentation of neurogenesis in the adult human brain in response to neuronal cell loss has not been demonstrated. This study was undertaken to investigate whether neurogenesis occurs in the subependymal layer (SEL) adjacent to the caudate nucleus in the human brain in response to neurodegeneration of the caudate nucleus in Huntington's disease (HD). Postmortem control and HD human brain tissue were examined by using the cell cycle marker proliferating cell nuclear antigen (PCNA), the neuronal marker beta III-tubulin, and the glial cell marker glial fibrillary acidic protein (GFAP). We observed a significant increase in cell proliferation in the SEL in HD compared with control brains. Within the HD group, the degree of cell proliferation increased with pathological severity and increasing CAG repeats in the HD gene. Most importantly, PCNA+ cells were shown to coexpress beta III-tubulin or GFAP, demonstrating the generation of neurons and glial cells in the SEL of the diseased human brain. Our results provide evidence of increased progenitor cell proliferation and neurogenesis in the diseased adult human brain and further indicate the regenerative potential of the human brain.

N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL) is increased in cerebrospinal fluid of patients with Huntington's disease.

Pathological-length polyglutamine (Q(n)) expansions, such as those that occur in the huntingtin protein (htt) in Huntington's disease (HD), are excellent substrates for tissue transglutaminase in vitro, and transglutaminase activity is increased in post-mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patients in vivo is scarce. We now report that levels of N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL)--a 'marker' isodipeptide produced by the transglutaminase reaction--are elevated in the CSF of HD patients (708 +/- 41 pmol/mL, SEM, n = 36) vs. control CSF (228 +/- 36, n = 27); p < 0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brain in vivo.

Hints about Huntington's disease

Scientists from Johns Hopkins University have defined a mechanism linking abnormal huntingtin protein to neuronal cell death. Mutant huntingtin, with an expanded polyglutamine stretch, interacts with CREB binding protein (CBP), recruits it into cellular aggregates, and prevents it from participating as a co-activator in CREB-mediated transcription. This nuclear depletion and aggregate enrichment of CBP was observed in Huntington's disease (HD) culture and transgenic mice models as well as in human postmortem HD brain tissue. Furthermore, the cell death seen with mutant huntingtin expression in cells grown in culture is decreased following overexpression of CBP, supporting the current hypothesis that the toxic gain-of-function seen with mutant huntingtin involves transcriptional disruption of cell-survival programs. The full paper is published in the March 23 issue of Science. LO

N-acetylaspartylglutamate, N-acetylaspartate, and N-acetylated alpha-linked acidic dipeptidase in human brain and their alterations in Huntington and Alzheimer's diseases.

There is mounting evidence, primarily from research in experimental animals, that the dipeptide N-acetylaspartylglutamate (NAAG) and its metabolic enzyme, N-acetylated alpha-linked acid dipeptidase (NAALADase), are involved in glutamatergic neurotransmission. Previous studies in neuropsychiatric disorders associated with the dysregulation of glutamatergic neurotransmission, such as schizophrenia, seizure disorders, and amyotrophic lateral sclerosis (ALS), have revealed region-specific alterations in the levels of NAAG and in the activity of NAALADase. To establish better the cellular localization of these and related parameters in human brain, we have examined their alterations in two well-characterized selective neurodengenerative disorders, Huntington Disease (HD) and Alzheimer Disease (AD). Brain regions from postmortem controls and HD- or AD-affected individuals were assayed to determine the activity of NAALADase as well as the levels of NAAG, N-acetylaspartate (NAA), and several amino acids. The relationships between changes in these neurochemical parameters and changes in neuronal and glial cell density were determined. The present report demonstrates that the decreases in the levels of NAAG and NAA and in the activity of NAALADase in AD and HD brain correlate primarily with neuronal loss. By inference, the results suggest that NAAG and NAA have primarily a neuronal localization in human brain and that there is a close relationship between NAAG and the dipeptidase NAALADase in populations of affected neurons.