Brains Of Huntington's Disease Patients Research Articles

Blocking acid-sensing ion channel 1 alleviates Huntington's disease pathology via an ubiquitin-proteasome system-dependent mechanism

Huntington's disease (HD) is a fatal neurodegenerative disorder. Despite a tremendous effort to develop therapeutic tools in several HD models, there is no effective cure at present. Acidosis has been observed previously in cellular and in in vivo models as well as in the brains of HD patients. Here we challenged HD models with amiloride (Ami) derivative benzamil (Ben), a chemical agent used to rescue acid-sensing ion channel (ASIC)-dependent acidotoxicity, to examine whether chronic acidosis is an important part of the HD pathomechanism and whether these drugs could be used as novel therapeutic agents. Ben markedly reduced the huntingtin-polyglutamine (htt-polyQ) aggregation in an inducible cellular system, and the therapeutic value of Ben was successfully recapitulated in the R6/2 animal model of HD. To reveal the mechanism of action, Ben was found to be able to alleviate the inhibition of the ubiquitin-proteasome system (UPS) activity, resulting in enhanced degradation of soluble htt-polyQ specifically in its pathological range. More importantly, we were able to demonstrate that blocking the expression of a specific isoform of ASIC (asic1a), one of the many molecular targets of Ben, led to an enhancement of UPS activity and this blockade also decreased htt-polyQ aggregation in the striatum of R6/2 mice. In conclusion, we believe that chemical compounds that target ASIC1a or pharmacological alleviation of UPS inhibition would be an effective and promising approach to combat HD and other polyQ-related disorders.

Inhibition of neurite outgrowth and promotion of cell death by cytoplasmic soluble mutant huntingtin stably transfected in mouse neuroblastoma cells

Huntington's disease (HD) is an autosomal dominant inheritable neurodegenerative disorder caused by expansion of a polyglutamine repeat in the amino-terminal region of huntingtin. Polyglutamine expansion causes mutant huntingtin to aggregate and accumulate in the nuclei and cytoplasm of neurons. The aggregated amino-terminal fragments of mutant huntingtin are toxic to neuronal cells and may be involved in the neurodegeneration in HD patient brains. Although nuclear mutant huntingtin has been found to affect gene expression, the effect of cytoplasmic mutant huntingtin remains to be investigated. We established stably transfected mouse neuroblastoma (N2a) cells that express soluble amino-terminal fragments of huntingtin containing 20 (20Q) or 150 (150Q) glutamine repeats. In these stable cell lines, both 20Q and 150Q are diffusely distributed in the cytoplasm without aggregate formation. However, the stable 150Q cells are deficient in neurite outgrowth. Compared with wild-type N2a cells and cells stably expressing 20Q, stable 150Q cells also have decreased viability and are more susceptible to apoptotic stimulation. These findings suggest that the cytoplasmic soluble mutant huntingtin is also toxic to cells.

Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens

Many diverse human diseases are associated with protein aggregation in ordered fibrillar structures called amyloid. Amyloid formation may mediate aberrant protein interactions that culminate in neurodegeneration in Alzheimer, Huntington, and Parkinson diseases and in prion encephalopathies. Studies of protein aggregation in the brain are hampered by limitations in imaging techniques and often require invasive methods that can only be performed postmortem. Here we describe transgenic mice in which aggregation-prone proteins that cause Huntington and Parkinson disease are expressed in the ocular lens. Expression of a mutant huntingtin fragment or alpha-synuclein in the lens leads to protein aggregation and cataract formation, which can be monitored in real time by noninvasive, highly sensitive optical techniques. Expression of a mutant huntingtin fragment in mice lacking the major lens chaperone, alphaB-crystallin, markedly accelerated the onset and severity of aggregation, demonstrating that the endogenous chaperone activity of alphaB-crystallin suppresses aggregation in vivo. These novel mouse models will facilitate the characterization of protein aggregation in vivo and are being used in efficient and economical screens for chemical and genetic modifiers of disease-relevant protein aggregation.

Mitogen‐ and stress‐activated protein kinase‐1 deficiency is involved in expanded‐huntingtin‐induced transcriptional dysregulation and striatal death

Huntington's disease (HD) is a neurodegenerative disorder due to an abnormal polyglutamine expansion in the N-terminal region of huntingtin protein (Exp-Htt). This expansion causes protein aggregation and neuronal dysfunction and death. Transcriptional dysregulation due to Exp-Htt participates in neuronal death in HD. Here, using the R6/2 transgenic mouse model of HD, we identified a new molecular alteration that could account for gene dysregulation in these mice. Despite a nuclear activation of the mitogen-activated protein kinase/extracellular regulated kinase (ERK) along with Elk-1 and cAMP responsive element binding, two transcription factors involved in c-Fos transcription, we failed to detect any histone H3 phosphorylation, which is expected after nuclear ERK activation. Accordingly, we found in the striatum of these mice a deficiency of mitogen- and stress-activated kinase-1 (MSK-1), a kinase downstream ERK, critically involved in H3 phosphorylation and c-Fos induction. We extended this observation to Exp-Htt-expressing striatal neurons and postmortem brains of HD patients. In vitro, knocking out MSK-1 expression potentiated Exp-Htt-induced striatal death. Its overexpression induced H3 phosphorylation and c-Fos expression and totally protected against striatal neurodegeneration induced by Exp-Htt. We propose that MSK-1 deficiency is involved in transcriptional dysregulation and striatal degeneration. Restoration of its expression and activity may be a new therapeutic target in HD.

Time-lapse analysis of aggregate formation in an inducible PC12 cell model of Huntington's disease reveals time-dependent aggregate formation that transiently delays cell death

Huntington's disease (HD) is a progressive neurodegenerative disease caused by a CAG repeat expansion in the coding region of the HD gene. The translated polyglutamine expansion causes the formation of insoluble aggregates in the brains of HD patients and transgenic mouse models. However, the relationship between aggregate formation and neuropathology remains unknown. We used fluorescent protein tagging and live-cell time-lapse microscopy to study visible aggregate formation and its relationship to cell death in transgenic PC12 cells. We used cell lines expressing a fragment of huntingtin exon 1 with either 23 (wild type) or 74 (mutant) glutamines fused to enhanced green fluorescent protein under the control of an inducible promoter. Live cells were observed in real time after transgene induction for up to 96 h. We found that aggregate formation was time-dependent and predominantly nuclear in these cells. We followed inclusion formation in individual cells, examining the cells every 10 min for up to 48 h. This revealed new details of inclusion formation. Initial aggregate formation was rapid (often <1 h), but many (18–>48) h were needed to establish a final aggregate phenotype. Aggregates formed in a dynamic manner and were in constant motion within cell nuclei throughout their maturation. The formation of large aggregates occurred more frequently in cells that survived longer. However, aggregate size was not a good predictor of cell death, since cells could die with either large (>2 μm), small (<0.5 μm) or no visible aggregates. Cells that formed large aggregates survived longer than cells that formed small aggregates or no aggregates at all. However, the time taken for a cell to die decreased as a function of increasing size of final aggregate formed. Further, cells that formed aggregates earlier tended to die earlier. Together our data are compatible with a toxic role for aggregates/aggregation and support the ‘toxic precursor’ hypothesis. However, they also suggest that at some stages, the process of aggregate formation is cytoprotective.

Severe deficiency of the fatty acid amide hydrolase (FAAH) activity segregates with the Huntington's disease mutation in peripheral lymphocytes

The search for peripheral markers of neurodegenerative diseases aims at identifying molecules that could help in monitoring the effects of future therapeutics in easily accessible cells. Here we focused on the involvement of the endocannabinoid system in Huntington's disease (HD). We assayed peripheral lymphocytes from HD patients and healthy controls, and found that the activity of the fatty acid amide hydrolase (FAAH), the enzyme that degrades the endocannabinoid anandamide (AEA), was dramatically decreased (down to less than 10%) in HD compared to healthy subjects. Concomitantly, the endogenous levels of AEA were approximately 6-fold higher in HD versus healthy lymphocytes, while the other elements of the endocannabinoid system were not affected by HD. Low FAAH activity in HD lymphocytes was not due to down-regulation of protein expression, but rather to blockage of enzyme activity by a cytosolic and irreversible inhibitor. Finally, pre-HD patients showed defective FAAH activity, as did the brain of HD patients compared with healthy controls. Taken together, our data indicate that FAAH activity in lymphocytes mirrors some of the metabolic changes which take place in the brain, it is a measurable non-genetic peripheral marker that segregates with the HD mutation, and it might serve as a target to test chemicals active on the widespread toxic effects of the mutant protein.

Hsp27 overexpression in the R6/2 mouse model of Huntington's disease: chronic neurodegeneration does not induce Hsp27 activation

Huntington's disease (HD) is caused by an expanded polyglutamine tract in the huntingtin protein. Mitochondrial dysfunction and free radical damage occur in both R6/2 mice and HD patient brains and might play a role in disease pathogenesis. In cell culture systems, heat-shock protein 27 (Hsp27), a small molecular chaperone, suppresses mutant huntingtin-induced reactive oxygen species formation and cell death. To investigate this in vivo, we conducted an extensive phenotypic characterization of mice arising from a cross between R6/2 mice and Hsp27 transgenic mice but did not observe an improvement of the R6/2 phenotype. Hsp27 overexpression had no effect in reducing oxidative stress in the R6/2 brain, assessed by measuring striatal aconitase activity and protein carbonylation levels. Native protein gel analysis revealed that transgenic Hsp27 forms active, large oligomeric species in heat-shocked brain lysates, demonstrating that it is efficiently activated upon stress. In contrast, Hsp27 in double transgenic brains exists predominantly as a low molecular weight, inactive species. This suggests that Hsp27, which is otherwise activatable upon heat shock, remains inactive in the R6/2 model of chronic neurodegeneration. Hsp27 transgenics had been previously shown to be protected from acute stresses such as kainate administration, ischemia/reperfusion heart injury and neonatal nerve injury. Our study is the first to suggest a differential modulation of Hsp27 activation in vivo and, importantly, it illustrates the diverse effect of Hsp27 on acute versus chronic models of disease.

Structural Insights into the Specific Binding of Huntingtin Proline-Rich Region with the SH3 and WW Domains

The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

Cytotoxicity of a mutant huntingtin fragment in yeast involves early alterations in mitochondrial OXPHOS complexes II and III

Mitochondrial dysfunction may play an important role in the pathogenic mechanism of Huntington's disease (HD). However, the exact mechanism by which mutated huntingtin could cause bioenergetic dysfunction is still unknown. We have constructed a stable inducible yeast model of HD by expressing a human huntingtin fragment containing a mutant polyglutamine tract of 103Q fused to green fluorescent protein (GFP), and a control expressing a wild-type 25Q domain fused to GFP in a wild-type strain. We showed that in yeast cells expressing 103Q, cell respiration was progressively reduced after 4-6 h of induction with galactose, down to 50% of the control after 10 h of induction. The cell respiration defect results from an alteration in the function and amount of mitochondrial respiratory chain complex II+III, in congruency to data obtained from postmortem brain of HD patients and from toxin models. In our model, the production of reactive oxygen species (ROS) is significantly enhanced in cells expressing 103Q. Quenching of ROS with resveratrol partially prevents the cell respiration defect. Mitochondrial morphology and distribution were also altered in cells expressing 103Q, probably resulting from the interaction of aggregates with portions of the mitochondrial web and from a progressive disruption of the actin cytoskeleton. We propose a mechanism for mitochondrial dysfunction in our yeast model of HD in which the interactions of misfolded/aggregated polyglutamine domains with the mitochondrial and actin networks lead to disturbances in mitochondrial distribution and function and to increase in ROS production. Oxidative damage could preferentially affect the stability and function of enzymes containing iron-sulfur clusters such as complexes II and III. Our yeast model represents a very useful paradigm to study mitochondrial physiology alterations in the pathogenic mechanism of HD.

A Role for Glia in Huntington's Disease?

Huntington's disease (HD), a devastating neurodegenerative disorder caused by an expanded polyglutamine tract in the protein huntingtin (htt), is characterized by the preferential loss of medium-sized spiny neurons (MSNs) in the striatum. Noting that htt is found in glia, as well as in neurons, and that MSNs receive abundant glutamatergic input (making them vulnerable to excitotoxicity), Shin et al. investigated the possible role of glial dysfunction in contributing to the pathophysiology of HD. The authors used immunogold labeling and immunofluorescence analysis to demonstrate the existence of intranuclear aggregates of htt in glia in the brains of R6/2 mice (a transgenic mouse model of HD). Intranuclear aggregates of htt were also found in glia of a second mouse model of HD and in brains of HD patients. The prevalence of nuclear htt aggregates in R6/2 glia increased with age and correlated temporally with a decrease in the abundance of glutamate transporter 1 (GLT-1). Consistent with the decrease in GLT-1, R6/2 brain slices showed decreased dihydrokainate-inhibitable glutamate uptake compared with wild-type brain slices, and this difference became more prominent with age. Coculture with wild-type astrocytes attenuated signs of neuronal degeneration in neurons overexpressing mutant htt, whereas coculture with glia expressing mutant htt promoted the degeneration of wild-type neurons. Moreover, wild-type astrocytes were better than R6/2 astrocytes at protecting neurons from glutamate-mediated toxicity. Thus, the authors suggest that expression of mutant htt in glia may lead to decreased glutamate uptake and thereby contribute to excitotoxic neuronal death. J.-Y. Shin, Z.-H. Fang, Z.-X. Yu, C.-E. Wang, S.-H. Li, X.-J. Li, Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity. J. Cell Biol. 171 , 1001-1012 (2005). [Abstract] [Full Text]

Increased glutathione levels in cortical and striatal mitochondria of the R6/2 Huntington's disease mouse model

Huntington's disease (HD) is a progressive neurodegenerative disease characterized by a severe neuronal loss that occurs primarily in the neostriatum. It has been postulated that mitochondria dysfunction and oxidative stress may play significant roles in the etiology of the disease. Indeed, markers of oxidative stress damage have been detected in the brains of HD patients and in mouse models of HD. In this study, we evaluate the changes in the levels of the potent, endogenous antioxidant glutathione and enzymes involved in its metabolism or recycling in the cortex and striatum of an extensively studied HD mouse model (R6/2). In both cortex and striatum, the levels of cellular glutathione were not significantly different in the R6/2 mice when compared with littermate wild type controls. Remarkably, the levels of glutathione were significantly increased in mitochondria isolated from the cortex and striatum of R6/2 mice when compared with wild type control mice. This specific increase in the levels of glutathione in mitochondria suggests that a compensatory mechanism is induced in the R6/2 mice to protect against an increase in oxidative stress in mitochondria.

Huntingtin is cleaved by caspases in the cytoplasm and translocated to the nucleus via perinuclear sites in Huntington's disease patient lymphoblasts

Accumulation of mutant Huntingtin (Htt), especially the N-terminal-cleaved Htt, participates in the pathophysiology of Huntington's disease (HD). It is difficult to elucidate temporal properties of the translocation of "endogenous" Htt using autopsy HD patient brains. Thus, we examined the cell biology of "endogenous" Htt cleavage and nuclear translocation in cultured lymphoblasts of HD patients and controls. Apoptotic stimulation of lymphoblasts elicits caspase-dependent cleavage and selective nuclear translocation of N-terminal portions of Htt. Discrete clusters of the N-terminal Htt accumulate at unique perinuclear sites prior to nuclear translocation. Our findings suggest that caspase cleavage of Htt is cytoplasmic and precedes sorting to specific perinuclear sites followed by nuclear translocation in HD patient tissue.

Adenosine and glutamate extracellular concentrations and mitogen-activated protein kinases in the striatum of Huntington transgenic mice. Selective antagonism of adenosine A 2A receptors reduces transmitter outflow

The basal ganglia and deep layers of cerebral cortex neurodegeneration typically characterize the postmortem brain of Huntington disease (HD) patients. In this study, we employed 10- to 11-week-old transgenic HD mice (R6/2 line), in which the striatal adenosine extracellular levels, measured using the microdialysis technique, are significantly increased in comparison to wild-type mice. An increase in striatal adenosine is probably a precocious index of mitochondrial dysfunction that is described in both the postmortem brain of HD patients and transgenic mice striatal cells. The adenosine increase is matched by activation of the p38 mitogen-activated protein kinase (MAPK) in the striatal neurons of R6/2 mouse but not in the cortex. This result indicates that p38 MAPK is a correlate of striatal damage and suggests a role for p38 in the striatal neuron suffering and apoptosis described in this disease. The selective adenosine A 2A receptor antagonist SCH 58261, administered through microdialysis fiber into the striatum, significantly decreases the outflow of glutamate in R6/2 mice. Antagonism of adenosine A 2A receptors might be regarded as potentially useful in the treatment of this disease to control striatal excitotoxicity.

The serum- and glucocorticoid-induced kinase SGK inhibits mutant huntingtin-induced toxicity by phosphorylating serine 421 of huntingtin.

Huntington's disease (HD) is caused by abnormal polyglutamine (polyQ) expansion in the protein huntingtin. We have previously demonstrated the importance of the insulin-like growth factor I (IGF-1)/Akt pathway in HD. Indeed, upon IGF-1 activation, Akt phosphorylates polyQ-huntingtin at serine 421 and abrogates its toxicity. In addition, we have demonstrated that Akt is altered in the brain of HD patients. Here, we investigate the role of the serum- and glucocorticoid-induced kinase (SGK) in HD. We show that SGK phosphorylates huntingtin at serine 421 and that phosphorylation can protect striatal neurons against polyQ-huntingtin-induced toxicity. We find that SGK levels are increased in the brain of HD patients. Using a cellular model of HD, we demonstrate that the SGK dysregulation induced by polyQ-huntingtin occurs via the p38/MAPK pathway. Collectively, our results strongly suggest the involvement of SGK in HD and further imply that IGF-1 downstream signalling is a key transduction pathway that regulates the toxicity of huntingtin.

Inclusion formation in Huntington's disease R6/2 mouse muscle cultures.

Huntington's disease (HD) is an autosomal dominant disorder caused by an expansion in the number of glutamine repeats in the N-terminal region of the huntingtin protein. Nuclear and cytoplasmic aggregates of the N-terminal portion of huntingtin have been found in the brains of HD patients and the brains and non-neuronal tissues of the R6/2 HD transgenic mouse. We have cultured myoblasts and myotubes from transgenic R6/2 mice and littermate controls to investigate the formation of these inclusions in post mitotic cells. Huntingtin immunoreactivity was intense in differentiating, desmin positive myoblasts and myotubes from both control and R6/2 mice suggesting that it may play a role in myotube differentiation. Following differentiation huntingtin and ubiquitin positive aggregates were observed in R6/2 but not control cultures. After 3 weeks in differentiation medium cytoplasmic huntingtin and ubiquitin immunoreactive aggregates were observed in non-myotube cells, while nuclear huntingtin aggregates were seen in a proportion of myotubes after 6 weeks. Growth in the absence of serum resulted in a marked increase in the number of R6/2 myotubes containing nuclear inclusions after 6 weeks demonstrating that environmental factors influenced huntingtin aggregate formation in these cells. Consequently, cultured myotubes from R6/2 mice may be a useful post mitotic cell culture model to study both the biochemical consequences of huntingtin aggregates and the factors that may influence aggregate formation.

Antisense downregulation of mutant huntingtin in a cell model.

Huntington's disease (HD) is an inherited neurodegenerative disorder which is caused by an expansion of a CAG repeat sequence in the HD gene. The repeat encodes an expanded polyglutamine tract in the protein huntingtin. The still unknown pathological mechanisms leading to death of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique. NT2 precursor cells and differentiated postmitotic NT2-N neurons, respectively, were transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection or by addition to the culture medium. Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression of the fusion protein and/or suppression of the aggregate formation in both cell types. In the NT2 cells the antisense effect was dependent on the way of administration of the oligo. The PS-antisense oligo is effective in downregulation of mutant huntingtin, and the reduction of aggregate formation is a sensitive biological marker. The findings suggest that antisense knockdown of huntingtin could be a useful strategy for treatment of HD, and could also be suitable for studies of the normal and pathological function of huntingtin in different cellular model systems.

The bacterial chaperonin GroEL requires GroES to reduce aggregation and cell death in a COS-7 cell model of Huntington's disease

Huntington's disease (HD) is caused by expansions of more than 35 CAG repeats in the HD gene. These repeats are translated into a long polyglutamine tract that confers a deleterious gain-of-function on the mutant protein. Intraneuronal inclusions comprising mutant huntingtin are found in HD patient brains. Here we show that the bacterial chaperonin GroEL can reduce aggregation of mutant huntingtin in COS-7 cells and requires GroES for efficient activity, analogous to what has been described in bacteria. The reduction in aggregation of mutant huntingtin by GroEL/GroES was associated with protection against polyglutamine-induced cell death.

Alleviation of motor hyperactivity and neurochemical deficits by endocannabinoid uptake inhibition in a rat model of Huntington's disease.

Recent studies have demonstrated a loss of cannabinoid CB1 receptors in the postmortem basal ganglia of patients affected by Huntington's disease (HD) and in transgenic mouse models for this disease. These studies have led to the notion that substances that increase the endocannabinoid activity, such as receptor agonists or inhibitors of endocannabinoid uptake and/or metabolism, might be useful in the treatment of hyperkinetic symptoms of this disease. In the present study, we employed a rat model of HD generated by bilateral intrastriatal injections of 3-nitropropionic acid (3-NP), a toxin that selectively damages striatal GABAergic efferent neurons. These rats exhibited biphasic motor disturbances, with an early (1-2 weeks) hyperactivity followed by a late (3-4 weeks) motor depression. Analysis of GABA, dopamine, and their related enzymes, glutamic acid decarboxylase and tyrosine hydroxylase, in the basal ganglia proved marked decreases compatible with the motor hyperkinesia. In addition, mRNA levels for CB1 receptor, neuronal-specific enolase, proenkephalin, and substance P decreased in the caudate-putamen of 3-NP-injected rats. There were also reductions in CB1 receptor binding in the caudate putamen, the globus pallidus, and, to a lesser extent, the substantia nigra. By contrast, mRNA levels for tyrosine hydroxylase in the substantia nigra remained unaffected. Interestingly, the administration of AM404, an inhibitor of endocannabinoid uptake, to 3-NP-injected rats attenuated motor disturbances observed in the early phase of hyperactivity. Administration of AM404 also tended to induce recovery from the neurochemical deficits caused by the toxin in GABA and dopamine indices in the basal ganglia. In summary, morphological, behavioral, and biochemical changes observed in rats intrastriatally lesioned with 3-NP acid were compatible with a profound degeneration of striatal efferent GABAergic neurons, similar to that occurring in the brain of HD patients. As expected, a loss of CB1 receptors was evident in the basal ganglia of these rats. However, the administration of substances that increase endocannabinoid activity, by inhibiting the uptake process, allowed an activation of the remaining population of CB1 receptors, resulting in a significant improvement of motor disturbances and neurochemical deficits. These observations might be relevant to the treatment of hyperkinetic symptoms in HD, a human disorder with unsatisfactory symptomatic treatment for most patients.

An open and shut case of neurodegeneration

Progressive neurodegenerative diseases typified by expansions of polyglutamine (polyQ) repeats tend to have a relatively focused etiology, with an emphasis on post-translational protein dysregulation underlying the pathology. Huntington's disease (HD) is an inherited late-onset polyQ disorder, in which the offending mutant protein, huntingtin (Htt), is susceptible to proteolytic cleavage, phosphorylation and/or aggregation during the disease progression; somehow, these changes relate to the death of neurons. An additional conductor of the HD orchestra has been proposed; evidence also implicates transcriptional dysregulation as an important component in disease progression. It has been postulated that aggregates of Htt might alter basal transcriptional levels of key housekeeping proteins and, thereby, compromise the function and health of neurons. Important insight into how this might occur has now been provided in a study showing Htt interaction with, and inhibition of, histone acetyltransferases. Histone acetylation is associated with an open, transcriptionally active chromatin state, whereas deacetylation is correlated with a closed chromatin state and gene repression or silencing. Steffan et al. [1xHistone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Steffan, J.S. et al. Nature. 2001; 413: 739–743Crossref | PubMed | Scopus (831)See all References[1] now show that deacetylase activity contributes to the neurodegeneration stimulated by Htt with polyQ repeats and deacetylase (HDAC) inhibitors can slow neurodegeneration in an invertebrate model of HD.Previous findings had indicated that mutant Htt interacts directly with the CREB-binding protein (CBP), which contains an acetyltransferase domain and is a component of a protein complex that acts on many promoters to activate genes. A protein fragment corresponding to exon 1 of Htt, containing the polyQ repeats (Httex1p), causes HD-like pathology in mice and inhibits, through direct binding, the acetyltransferase activity of a number of proteins. The interaction is not dependent on the polyQ regions found naturally in the acetyltransferases, but is dependent on the length of the polyQ sequence found in the Httex1p fragment, suggesting that a targeted, gain of inhibitory function is endowed upon Httex1p by polyQ extension. To verify the direct acetyltransferase inhibitory activity of Httex1p in cells, two approaches were taken. First, cells were transfected with polyQ Httex1p and the degree of histone acetylation, but not histone protein expression, was shown to be decreased, an effect that was reversed when HDAC inhibitors were applied also. Second, Drosophila photoreceptor neurons engineered to express either polyQ Httex1p or polyQ peptides in vivo degenerate progressively, but display a dose-dependent increase in vitality when HDAC inhibitors were also fed to flies. Inhibitor-treated adult flies exhibited a repressed early-onset death; early-onset death is observed following polyQ Httex1p expression. A partial loss of function allele for the gene Sin3A, which encodes a protein component of HDAC complexes in Drosophila, also increased the viability of and reduced neurodegeneration in polyQ-Httex1p-containing flies.Studies have shown that CBP and other transcriptional regulatory proteins are sequestered in cytoplasmic and nuclear aggregates, both in HD mouse models and in the brains of HD patients. Locking away the key architects of higher order chromatin design and gene transcription probably lead to a repression of those genes required normally to keep neurons functional and viable. Time will tell whether HDAC inhibitors will prove to be possible pharmacological sources of novel neurodegeneration therapeutics. When administered to flies already demonstrating neurodegeneration, the inhibitors arrest further degeneration markedly, indicating an effect on the cause, rather than just the symptoms, of the disease. Roll on the rodent model!

VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration.

Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders.