Edward C. Stack

Chronology of behavioral symptoms and neuropathological sequela in R6/2 Huntington's disease transgenic mice.

Genetic murine models play an important role in the study of human neurological disorders by providing accurate and experimentally accessible systems to study pathogenesis and to test potential therapeutic treatments. One of the most widely employed models of Huntingtons disease (HD) is the R6/2 transgenic mouse. To characterize this model further, we have performed behavioral and neuropathological analyses that provide a foundation for the use of R6/2 mice in preclinical therapeutic trials. Behavioral analyses of the R6/2 mouse reveal age‐related impairments in dystonic movements, motor performance, grip strength, and body weight that progressively worsen until death. Significant neuropathological sequela, identified as increasing marked reductions in brain weight, are present from 30 days, whereas decreased brain volume is present from 60 days and decreased neostriatal volume and striatal neuron area, with a concomitant reduction in striatal neuron number, are present at 90 days of age. Huntingtin‐positive aggregates are present at postnatal day 1 and increase in number and size with age. Our findings suggest that the R6/2 HD model exhibits a progressive HD‐like behavioral and neuropathological phenotype that more closely corresponds to human HD than previously believed, providing further assurance that the R6/2 mouse is an appropriate model for testing potential therapies for HD. J. Comp. Neurol. 490:354–370, 2005.

Evidence of oxidant damage in Huntington's disease: translational strategies using antioxidants.

Huntingtons disease (HD) is an autosomal dominant inherited neurodegenerative disorder characterized by progressive motor dysfunction, emotional disturbances, dementia, and weight loss. It is caused by an expanded trinucleotide CAG repeat in the gene coding for the protein, huntingtin. Although no one specific interaction of mutant huntingtin has been suggested to be the pathologic trigger, a large body of evidence suggests that, in both the human condition and in HD mice, oxidative stress may play a role in the pathogenesis of HD. Increased levels of oxidative damage products, including protein nitration, lipid peroxidation, DNA oxidation, and exacerbated lipofuscin accumulation, occur in HD. Strong evidence exists for early oxidative stress in HD, coupled with mitochondrial dysfunction, each exacerbating the other and leading to an energy deficit. If oxidative damage plays a role in HD, then therapeutic strategies that reduce reactive oxygen species may ameliorate the neurodegenerative process. Two such strategies, using coenzyme Q10 and creatine, have been proposed. Although each agent has had limited efficacy in HD patients, the optimal therapeutic dose may have been underestimated. High‐dose coenzyme Q10 and creatine are safe and tolerable in HD patients and are currently under investigation. In addition, there are parallels in reducing markers of oxidative stress in both HD mice and HD patients after treatment. It is likely that high‐dose coenzyme Q10, creatine, or both agents, will represent a cornerstone defense in ameliorating the progression of HD.

Genetic and pharmacological inactivation of the adenosine A2A receptor attenuates 3‐nitropropionic acid‐induced striatal damage

Adenosine A2A receptor (A2AR) antagonism attenuates 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced dopaminergic neurodegeneration and quinolinic acid‐induced excitotoxicity in the neostriatum. As A2ARs are enriched in striatum, we investigated the effect of genetic and pharmacological A2A inactivation on striatal damage produced by the mitochondrial complex II inhibitor 3‐nitropriopionic acid (3‐NP). 3‐NP was administered to A2AR knockout (KO) and wild‐type (WT) littermate mice over 5 days. Bilateral striatal lesions were analyzed from serial brain tissue sections. Whereas all of the 3‐NP‐treated WT mice (C57BL/6 genetic background) had bilateral striatal lesions, only one of eight of the 3‐NP‐treated A2AR KO mice had detectable striatal lesions. Similar attenuation of 3‐NP‐induced striatal damage was observed in A2AR KO mice in a 129‐Steel background. In addition, the effect of pharmacological antagonism on 3‐NP‐induced striatal neurotoxicity was tested by pre‐treatment of C57Bl/6 mice with the A2AR antagonist 8‐(3‐chlorostyryl) caffeine (CSC). Although bilateral striatal lesions were observed in all mice treated either with 3‐NP alone or 3‐NP plus vehicle, there were no demonstrable striatal lesions in mice treated with CSC (5 mg/kg) plus 3‐NP and in five of six mice treated with CSC (20 mg/kg) plus 3‐NP. We conclude that both genetic and pharmacological inactivation of the A2AR attenuates striatal neurotoxicity produced by 3‐NP. Since the clinical and neuropathological features of 3‐NP‐induced striatal damage resemble those observed in Huntingtons disease, the results suggest that A2AR antagonism may be a potential therapeutic strategy in Huntingtons disease patients.

Combined riluzole and sodium phenylbutyrate therapy in transgenic amyotrophic lateral sclerosis mice

Recent evidence suggests that transcriptional dysregulation may play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS). The histone deacetylase inhibitor, sodium phenylbutyrate (NaPB), is neuroprotective and corrects aberrant gene transcription in ALS mice and has recently been shown to be safe and tolerable in ALS patients while improving hypoacetylation. Since many patients are already on riluzole, it is important to ensure that any proposed therapy does not result in negative synergy with riluzole. The combined treatment of riluzole and NaPB significantly extended survival and improved both the clinical and neuropathological phenotypes in G93A transgenic ALS mice beyond either agent alone. Combination therapy increased survival by 21.5%, compared to the separate administration of riluzole (7.5%) and NaPB (12.8%), while improving both body weight loss and grip strength. The data show that the combined treatment was synergistic. In addition, riluzole/NaPB treatment ameliorated gross lumbar and ventral horn atrophy, attenuated lumbar ventral horn neuronal cell death, and decreased reactive astrogliosis. Riluzole/NaPB administration increased acetylation at H4 and increased NF-κB p50 translocation to the nucleus in G93A mice, consistent with a therapeutic effect. These data suggest that NaPB may not interfere with the pharmacologic action of riluzole in ALS patients.

RETROMER DISRUPTION PROMOTES AMYLOIDOGENIC APP PROCESSING

Retromer deficiency has been implicated in sporadic AD and animals deficient in retromer components exhibit pronounced neurodegeneration. Because retromer performs retrograde transport from the endosome to the Golgi apparatus and neuronal Aβ is found in late endosomal compartments, we speculated that retromer malfunction might enhance amyloidogenic APP processing by promoting interactions between APP and secretase enzymes in late endosomes. We have evaluated changes in amyloid precursor protein (APP) processing and trafficking as a result of disrupted retromer activity by knockdown of Vps35, a vacuolar sorting protein that is an essential component of the retromer complex. Knocking down retromer activity produced no change in the quantity or cellular distribution of total cellular APP and had no affect on internalization of cell-surface APP. Retromer deficiency did, however, increase the ratio of secreted Aβ42:Aβ40 in HEK-293 cells over-expressing APP695, due primarily to a decrease in Aβ40 secretion. Recent studies suggest that the retromer-trafficked protein, Wntless, is secreted at the synapse in exosome vesicles and that these same vesicles contain Aβ. We therefore hypothesized that retromer deficiency may be associated with altered exosomal secretion of APP and/or secretase fragments. Holo-APP, Presenilin and APP C-terminal fragments were detected in exosomal vesicles secreted from HEK-293 cells. Levels of total APP C-terminal fragments were significantly increased in exosomes secreted by retromer deficient cells. These data suggest that reduced retromer activity can mimic the effects of familial AD Presenilin mutations on APP processing and promote export of amyloidogenic APP derivatives.

IDENTIFICATION OF CHOLINERGIC AND NON-CHOLINERGIC NEURONS IN THE PONS EXPRESSING PHOSPHORYLATED CYCLIC ADENOSINE MONOPHOSPHATE RESPONSE ELEMENT-BINDING PROTEIN AS A FUNCTION OF RAPID EYE MOVEMENT SLEEP

Recent studies have shown that in the pedunculopontine tegmental nucleus (PPT), increased neuronal activity and kainate receptor-mediated activation of intracellular protein kinase A (PKA) are important physiological and molecular steps for the generation of rapid eye movement (REM) sleep. In the present study performed on rats, phosphorylated cyclic AMP response element-binding protein (pCREB) immunostaining was used as a marker for increased intracellular PKA activation and as a reflection of increased neuronal activity. To identify whether activated cells were either cholinergic or noncholinergic, the PPT and laterodorsal tegmental nucleus (LDT) cells were immunostained for choline acetyltransferase (ChAT) in combination with pCREB or c-Fos. The results demonstrated that during high rapid eye movement sleep (HR, approximately 27%), significantly higher numbers of cells expressed pCREB and c-Fos in the PPT, of which 95% of pCREB-expressing cells were ChAT-positive. With HR, the numbers of pCREB-positive cells were also significantly higher in the medial pontine reticular formation (mPRF), pontine reticular nucleus oral (PnO), and dorsal subcoeruleus nucleus (SubCD) but very few in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). Conversely, with low rapid eye movement sleep (LR, approximately 2%), the numbers of pCREB expressing cells were very few in the PPT, mPRF, PnO, and SubCD but significantly higher in the LC and DRN. The results of regression analyses revealed significant positive relationships between the total percentages of REM sleep and numbers of ChAT+/pCREB+ (Rsqr=0.98) cells in the PPT and pCREB+ cells in the mPRF (Rsqr=0.88), PnO (Rsqr=0.87), and SubCD (Rsqr=0.84); whereas significantly negative relationships were associated with the pCREB+ cells in the LC (Rsqr=0.70) and DRN (Rsqr=0.60). These results provide evidence supporting the hypothesis that during REM sleep, the PPT cholinergic neurons are active, whereas the LC and DRN neurons are inactive. More importantly, the regression analysis indicated that pCREB activation in approximately 98% of PPT cholinergic neurons, was caused by REM sleep. Moreover the results indicate that during REM sleep, PPT intracellular PKA activation and a transcriptional cascade involving pCREB occur exclusively in the cholinergic neurons.

Huntington's disease: progress and potential in the field

While the first description of Huntingtons disease was reported over a century ago, no therapy exists that can halt or ameliorate the inexorable disease progression. Tremendous progress, however, has been made in significantly broadening the understanding of pathogenic mechanisms in this neurological disorder that may eventually lead to successful treatment strategies. Huntingtons disease is caused by the expansion of a CAG repeat in the huntingtin gene, which results in the expression of a mutant form of the protein that is toxic to neurons. Several mechanisms have been identified in mediating this toxicity, such as protein aggregation, mitochondrial dysfunction, oxidative stress, transcriptional dysregulation, aberrant apoptosis, altered proteosomal function and excitotoxicity. With increasing understanding of each of these pathogenic mechanisms, therapeutic strategies have attempted to target specific aspects of each. There have been many encouraging reports of preclinical efficacy in transgenic Huntingtons disease mice, from which a number have been extended to human clinical trials with some success. This review focuses on these studies and the compounds that hold promise for treating human Huntingtons disease.

A novel role for calcium/calmodulin kinase II within the brainstem pedunculopontine tegmentum for the regulation of wakefulness and rapid eye movement sleep

J. Neurochem. (2010) 112, 271–281.