Steven M. Hersch

Ataxin-1 Nuclear Localization and Aggregation: Role in Polyglutamine-Induced Disease in SCA1 Transgenic Mice

Transgenic mice carrying the spinocerebellar ataxia type 1 (SCA1) gene, a polyglutamine neurodegenerative disorder, develop ataxia with ataxin-1 localized to aggregates within cerebellar Purkinje cells nuclei. To examine the importance of nuclear localization and aggregation in pathogenesis, mice expressing ataxin-1[82] with a mutated NLS were established. These mice did not develop disease, demonstrating that nuclear localization is critical for pathogenesis. In a second series of transgenic mice, ataxin-1[77] containing a deletion within the self-association region was expressed within Purkinje cells nuclei. These mice developed ataxia and Purkinje cell pathology similar to the original SCA1 mice. However, no evidence of nuclear ataxin-1 aggregates was found. Thus, although nuclear localization of ataxin-1 is necessary, nuclear aggregation of ataxin-1 is not required to initiate pathogenesis in transgenic mice.

Regional and progressive thinning of the cortical ribbon in Huntington’s disease

BackgroundHuntington’s disease (HD) is a fatal and progressive neurodegenerative disease that is accompanied by involuntary movements, cognitive dysfunction, and psychiatric symptoms. Although progressive striatal degeneration is known to occur, little is known about how the disease affects the cortex, including which cortical regions are affected, how degeneration proceeds, and the relationship of the cortical degeneration to clinical symptoms. The cortex has been difficult to study in neurodegenerative diseases primarily because of its complex folding patterns and regional variability; however, an understanding of how the cortex is affected by the disease may provide important new insights into it. MethodsNovel automated surface reconstruction and high-resolution MR images of 11 patients with HD and 13 age-matched subjects were used to obtain cortical thickness measurements. The same analyses were performed on two postmortem brains to validate these methods. ResultsRegionally specific heterogeneous thinning of the cortical ribbon was found in subjects with HD. Thinning occurred early, differed among patients in different clinical stages of disease, and appeared to proceed from posterior to anterior cortical regions with disease progression. The sensorimotor region was statistically most affected. Measurements performed on MR images of autopsy brains analyzed similarly were within 0.25 mm of those obtained using traditional neuropathologic methods and were statistically indistinguishable. ConclusionsThe authors propose that the cortex degenerates early in disease and that regionally selective cortical degeneration may explain the heterogeneity of clinical expression in HD. These measures might provide a sensitive prospective surrogate marker for clinical trials of neuroprotective medications.

A YAC Mouse Model for Huntington’s Disease with Full-Length Mutant Huntingtin, Cytoplasmic Toxicity, and Selective Striatal Neurodegeneration

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntingtons disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.

Nuclear and Neuropil Aggregates in Huntington’s Disease: Relationship to Neuropathology

The data we report in this study concern the types, location, numbers, forms, and composition of microscopic huntingtin aggregates in brain tissues from humans with different grades of Huntington’s disease (HD). We have developed a fusion protein antibody against the first 256 amino acids that preferentially recognizes aggregated huntingtin and labels many more aggregates in neuronal nuclei, perikarya, and processes in human brain than have been described previously. Using this antibody and human brain tissue ranging from presymptomatic to grade 4, we have compared the numbers and locations of nuclear and neuropil aggregates with the known patterns of neuronal death in HD. We show that neuropil aggregates are much more common than nuclear aggregates and can be present in large numbers before the onset of clinical symptoms. There are also many more aggregates in cortex than in striatum, where they are actually uncommon. Although the striatum is the most affected region in HD, only 1–4% of striatal neurons in all grades of HD have nuclear aggregates. Neuropil aggregates, which we have identified by electron microscopy to occur in dendrites and dendritic spines, could play a role in the known dendritic pathology that occurs in HD. Aggregates increase in size in advanced grades, suggesting that they may persist in neurons that are more likely to survive. Ubiquitination is apparent in only a subset of aggregates, suggesting that ubiquitin-mediated proteolysis of aggregates may be late or variable.

Immunocytochemical localization of D1 and D2 dopamine receptors in the basal ganglia of the rat: Light and electron microscopy

The modulatory actions of dopamine on the flow of cortical information through the basal ganglia are mediated mainly through two subtypes of receptors, the D1 and D2 receptors. In order to examine the precise cellular and subcellular location of these receptors, immunocytochemistry using subtype specific antibodies was performed on sections of rat basal ganglia at both the light and electron microscopic levels. Both peroxidase and pre-embedding immunogold methods were utilized. Immunoreactivity for both D1 and D2 receptors was most abundant in the neostriatum where it was mainly contained within spiny dendrites and in perikarya. Although some of the immunoreactive perikarya had characteristics of interneurons, most were identified as medium-sized spiny neurons. Immunoreactivity for D1 receptor but not D2 receptor was associated with the axons of the striatonigral pathway and axons and terminals in the substantia nigra pars reticulata and the entopeduncular nucleus. In contrast, D2 immunoreactivity but not D1 immunoreactivity was present in the dopaminergic neurons in the substantia nigra pars compacta and ventral pars reticulata. In the globus pallidus, little immunoreactivity for either D1 or D2 receptor was detected. At the subcellular level, D1 and D2 receptor immunoreactivity was found to be mainly associated with the internal surface of cell membranes. In dendrites and spines immunoreactivity was seen in contact with the membranes postsynaptic to terminals forming symmetrical synapses and less commonly, asymmetrical synapses. The morphological features and membrane specializations of the terminals forming symmetrical synapses are similar to those of dopaminergic terminals previously identified by immunocytochemistry for tyrosine hydroxylase. In addition to immunoreactivity associated with synapses, a high proportion of the immunoreactivity was also on membranes at non-synaptic sites. It is concluded that dopamine receptor immunoreactivity is mainly associated with spiny output neurons of the neostriatum and that there is a selective association of D1 receptors with the so-called direct pathway of information flow through the basal ganglia, i.e. the striatoentopeduncular and striatonigral pathways. Although there is an association of receptor immunoreactivity with afferent synaptic inputs a high proportion is located at extrasynaptic sites.

Huntingtin aggregates may not predict neuronal death in Huntington's disease

The mechanism by which polyglutamine expansion in Huntingtons disease (HD) results in selective neuronal degeneration remains unclear. We previously reported that the immunohistochemical distribution of N‐terminal huntingtin in HD does not correspond to the severity of neuropathology, such that significantly greater numbers of huntingtin aggregates are present within the cortex than in the striatum. We now show a dissociation between huntingtin aggregation and the selective pattern of striatal neuron loss observed in HD. Aggregate formation was predominantly observed in spared interneurons, with few or no aggregates found within vulnerable spiny striatal neurons. Multiple perikaryal aggregates were present in almost all cortical NADPH‐diaphorase neurons and in approximately 50% of the spared NADPH‐diaphorase striatal neurons from early grade HD cases. In severe grade HD patients, aggregates were more prominent as nuclear inclusions in NADPH‐diaphorase neurons, with less perikaryal and neuropil aggregation. In contrast, nuclear or perikaryal huntingtin aggregates were present in less than 4% of the vulnerable calbindin striatal neurons in all HD cases. These findings support the hypothesis that polyglutamine aggregation may not be a predictor of cell loss. Rather than a harbinger of neuronal death, mutant huntingtin aggregation may be a cytoprotective mechanism against polyglutamine‐induced neurotoxicity.

Ectopically Expressed CAG Repeats Cause Intranuclear Inclusions and a Progressive Late Onset Neurological Phenotype in the Mouse

The mutations responsible for several human neurodegenerative disorders are expansions of translated CAG repeats beyond a normal size range. To address the role of repeat context, we have introduced a 146-unit CAG repeat into the mouse hypoxanthine phosphoribosyltransferase gene (Hprt). Mutant mice express a form of the HPRT protein that contains a long polyglutamine repeat. These mice develop a phenotype similar to the human translated CAG repeat disorders. Repeat containing mice show a late onset neurological phenotype that progresses to premature death. Neuronal intranuclear inclusions are present in affected mice. Our results show that CAG repeats do not need to be located within one of the classic repeat disorder genes to have a neurotoxic effect.

Cerebral cortex and the clinical expression of Huntington's disease: complexity and heterogeneity

The clinical phenotype of Huntingtons disease (HD) is far more complex and variable than depictions of it as a progressive movement disorder dominated by neostriatal pathology represent. The availability of novel neuro-imaging methods has enabled us to evaluate cerebral cortical changes in HD, which we have found to occur early and to be topographically selective. What is less clear, however, is how these changes influence the clinical expression of the disease. In this study, we used a high-resolution surface based analysis of in vivo MRI data to measure cortical thickness in 33 individuals with HD, spanning the spectrum of disease and 22 age- and sex-matched controls. We found close relationships between specific functional and cognitive measures and topologically specific cortical regions. We also found that distinct motor phenotypes were associated with discrete patterns of cortical thinning. The selective topographical associations of cortical thinning with clinical features of HD suggest that we are not simply correlating global worsening with global cortical degeneration. Our results indicate that cortical involvement contributes to important symptoms, including those that have been ascribed primarily to the striatum, and that topologically selective changes in the cortex might explain much of the clinical heterogeneity found in HD. Additionally, a significant association between regional cortical thinning and total functional capacity, currently the leading primary outcome measure used in neuroprotection trials for HD, establishes cortical MRI morphometry as a potential biomarker of disease progression.

Caspase Cleavage of Mutant Huntingtin Precedes Neurodegeneration in Huntington's Disease

Huntingtons disease (HD) results from polyglutamine expansion in huntingtin (htt), a protein with several consensus caspase cleavage sites. Despite the identification of htt fragments in the brain, it has not been shown conclusively that htt is cleaved by caspases in vivo. Furthermore, no study has addressed when htt cleavage occurs with respect to the onset of neurodegeneration. Using antibodies that detect only caspase-cleaved htt, we demonstrate that htt is cleaved in vivo specifically at the caspase consensus site at amino acid 552. We detect caspase-cleaved htt in control human brain as well as in HD brains with early grade neuropathology, including one homozygote. Cleaved htt is also seen in wild-type and HD transgenic mouse brains before the onset of neurodegeneration. These results suggest that caspase cleavage of htt may be a normal physiological event. However, in HD, cleavage of mutant htt would release N-terminal fragments with the potential for increased toxicity and accumulation caused by the presence of the expanded polyglutamine tract. Furthermore, htt fragments were detected most abundantly in cortical projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD.

Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles

A cholinergic locus has recently been identified consisting of a unique mammalian genomic arrangement containing the genes for choline acetyltransferase (ChAT) and a putative vesicular acetylcholine transporter (VAChT). Although transcripts for ChAT and VAChT protein have been localized in cholinergic neurons, little is known about the encoded VAChT protein. Here we describe production of highly specific rabbit polyclonal antibodies, generated using a VAChT C- terminus/glutathione-S-transferase fusion protein, and immunological characterization of the native VAChT protein. These antibodies specifically recognized full-length recombinant VAChT expressed in transfected HeLa cells by Western blotting, with the prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single VAChT- immunoreactive band of approximately 70 kDa was predominant in known areas of cholinergic innervation, including striatum, cortex, hippocampus,and amygdala. Light microscopic immunocytochemistry revealed reaction product in cholinergic cell groups but not in noncholinergic areas. More significantly, immunoreactivity was also concentrated in axonal fibers in many regions known to receive prominent cholinergic innervation, such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem regions. Electron microscopy using immunoperoxidase revealed that VAChT was localized in axon terminals, and using more precise immunogold techniques, to synaptic vesicles. In VAChT-positive perikarya, the immunogold particles were localized to the cytoplasmic face of the Golgi complex. These findings confirm that VAChT protein is expressed uniquely in cholinergic neurons, concentrated in synaptic vesicles, and at least for the C terminus, topologically oriented as predicted by models.