Marian DiFiglia

Formation of Neuronal Intranuclear Inclusions Underlies the Neurological Dysfunction in Mice Transgenic for the HD Mutation

Huntingtons disease (HD) is one of an increasing number of human neurodegenerative disorders caused by a CAG/polyglutamine-repeat expansion. The mutation occurs in a gene of unknown function that is expressed in a wide range of tissues. The molecular mechanism responsible for the delayed onset, selective pattern of neuropathology, and cell death observed in HD has not been described. We have observed that mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to (CAG)156 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype. The appearance in transgenic mice of these inclusions, followed by characteristic morphological change within neuronal nuclei, is strikingly similar to nuclear abnormalities observed in biopsy material from HD patients.

Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons.

The gene defective in Huntingtons disease encodes a protein, huntingtin, with unknown function. Antisera generated against three separate regions of huntingtin identified a single high molecular weight protein of approximately 320 kDa on immunoblots of human neuroblastoma extracts. The same protein species was detected in human and rat cortex synaptosomes and in sucrose density gradients of vesicle-enriched fractions, where huntingtin immunoreactivity overlapped with the distribution of vesicle membrane proteins (SV2, transferrin receptor, and synaptophysin). Immunohistochemistry in human and rat brain revealed widespread cytoplasmic labeling of huntingtin within neurons, particularly cell bodies and dendrites, rather than the more selective pattern of axon terminal labeling characteristic of many vesicle-associated proteins. At the ultrastructural level, immunoreactivity in cortical neurons was detected in the matrix of the cytoplasm and around the membranes of the vesicles. The ubiquitous cytoplasmic distribution of huntingtin in neurons and its association with vesicles suggest that huntingtin may have a role in vesicle trafficking.

Excitotoxic injury of the neostriatum: a model for Huntington's disease

Intrastriatal lesions with excitatory amino acids mimic some of the neurochemical and neuropathological characteristics of Huntingtons disease (HD); this has led to the hypothesis that an endogenous excitotoxin may be involved in the disease. Recent advances in understanding the metabolic pathways of endogenous excitotoxins and the distribution and function of excitatory amino acid receptors have helped to refine the excitotoxin hypothesis, which is still inadequate to explain some aspects of the disease. However, as an experimental model for producing neuronal depletion in the neostriatum, excitotoxic injury has allowed the study of other neuronal characteristics of HD such as progressive atrophy and regeneration; it has also permitted extensive exploration of the anatomical and functional recovery induced by intrastriatal grafts. Moreover, adaptation of the rodent model to the non-human primate has enabled investigators to examine lesion-induced motor dysfunctions that are more comparable to those in HD. Thus, beyond its potential importance in the pathogenesis of HD, excitotoxic injury as an experimental tool promises to help further elucidate the pathological and functional alterations characteristic of the disease.

Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis

The Huntingtons disease (HD) mutation is a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). How neurons die in HD is unclear. Mutant N-htt aggregates in neurons in the HD brain; expression of mutant N-htt in vitro causes cell death. Other in vitro studies show that proteolysis by caspase 3 could be important in regulating mutant N-htt function, but there has been no direct evidence for caspase 3-cleaved N-htt fragments in brain. Here, we show that N-htt fragments consistent with the size produced by caspase 3 cleavage in vitro are resident in the cortex, striatum, and cerebellum of normal and adult onset HD brain and are similar in size to the fragments seen after exogenous expression of human huntingtin in mouse clonal striatal neurons. HD brain extracts treated with active caspase 3 had increased levels of N-htt fragments. Compared with the full-length huntingtin, the caspase 3-cleaved N-htt fragments, especially the mutant fragment, preferentially segregated with the membrane fraction. Partial proteolysis of the human caspase 3-cleaved N-htt fragment by calpain occurred in vitro and resulted in smaller N-terminal products; products of similar size appeared when mouse brain protein extracts were treated with calpain. Results support the idea that sequential proteolysis by caspase 3 and calpain may regulate huntingtin function at membranes and produce N-terminal mutant fragments that aggregate and cause cellular dysfunction in HD.

Autophagy and its possible roles in nervous system diseases, damage and repair.

Increased numbers of autophagosomes are seen in a variety of physiological and pathological states in the nervous system. In many cases, it is unclear if this phenomenon is the result of increased autophagic activity or decreased autophagosome-lysosome fusion. The functional significance of autophagy and its relationship to cell death in the nervous system is also poorly understood. In this review, we have considered these issues in the context of acute neuronal injury and a range of chronic neurodegenerative conditions, including the Lurcher mouse, Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases. While many issues remain unresolved, these conditions raise the possibility that autophagy can have either deleterious or protective effects depending on the specific situation and stage in the pathological process.

A Golgi study of neuronal types in the neostriatum of monkeys.

Examination of the nestriatum of monkeys prepared by the Golgi-Kopsch perfusion method revealed the presence of at least 6 neuronal types. The spiny type I is medium size with a high density of dendritic spines. The axon extends well beyond the dendritic field and gives off many collaterals. The spiny type II is either medium or large size, has long thick dendrites with a relatively low density of spines, and an axon similar to that of the previous type but with fever collaterals. The aspiny type I is medium size with varicose dendrites and a thin axon arborizing in the immediate vicinity of the soma. The aspiny type II is large, with many thick and thin varicose dendrites. The aspiny type III is medium size with smooth dendrites and an axon ramifying profusely within the dendritic field. The neurogliform cell is small with many branching processes. Findings indicate that the neostriatum has 2 distinct types of spiny neurons with long axons (spiny I and II), some of which may contribute to the efferent system. There are also 2 (aspiny I and III) or perhaps as many as 4 categories (aspiny I, II, III and neurogliform) of typical Golgi type II cells. Large neurons belong to 2 separate populations, one with dendritic spines and a long axon (large version of spiny II), and one with varicosities and presumably a short axon (aspiny II). A realistic interpretation of neurophysiologic data on the neostriatum must take into account all cell types instead of the current view of considering it as a pool of interneurons with few output cells.

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits

Huntingtons disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1–400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

Wild-Type and Mutant Huntingtins Function in Vesicle Trafficking in the Secretory and Endocytic Pathways☆

Huntingtin is a cytoplasmic protein that is found in neurons and somatic cells. In patients with Huntingtons disease (HD), the NH2-terminal region of huntingtin has an expanded polyglutamine tract. An abnormal protein interaction by mutant huntingtin has been proposed as a mechanism for HD pathogenesis. Huntingtin associates with vesicle membranes and interacts with proteins involved in vesicle trafficking. It is unclear where along vesicle transport pathways wild-type and mutant huntingtins are found and whether polyglutamine expansion affects this localization. To distinguish wild-type and mutant huntingtin, fibroblasts from normals and HD patients with two mutant alleles (homozygotes) were examined. Immunofluorescence confocal microscopy showed that mutant huntingtin localized with clathrin in membranes of the trans Golgi network and in clathrin-coated and noncoated endosomal vesicles in the cytoplasm and along plasma membranes. Separation of organelles in Nycodenz gradients showed that in normal and HD homozygote patient cells, huntingtin was present in membrane fractions enriched in clathrin. Similar results were obtained in fibroblasts from heterozyote juvenile HD patients who had a highly expanded polyglutamine tract in the HD allele. Western blot analysis of membrane fractions from rat brain showed that wild-type huntingtin was present in fractions that contained purified clathrin-coated membranes or a mixture of clathrin-coated and noncoated membranes. Electron microscopy of huntingtin immunoreactivity in rat brain revealed labeling along dendritic plasma membranes in association with clathrin-coated pits and clusters of noncoated endosomal vesicles 40-60 nm in diameter. These data suggest that wild-type and mutant huntingtin can influence vesicle transport in the secretory and endocytic pathways through associations with clathrin-coated vesicles.

The frequency and distribution of medium-sized neurons with indented nuclei in the primate and rodent neostriatum

Nuclear morphology is known to distinguish two classes of medium-sized neurons in the neostriatum, spiny projection neurons which have an unindented nucleus and aspiny interneurons which exhibit enfoldings of their nuclear envelope. The frequency and distribution of medium-sized neostriatal neurons with indented nuclei were examined in the light microscope in semi-thick sections. Results showed that the proportion of neostriatal neurons with nuclear indentations was greater in the monkey (23%) than in the mouse and rat (4-5%). Also, nuclear enfoldings were found in more neurons of the monkey caudate than putamen, whereas in the rodent regional differences were not observed. Findings suggest that aspiny interneurons play a greater role in the integrative and functional organization of the neostriatum in the primate than in the rodent brain.

Five siRNAs Targeting Three SNPs May Provide Therapy for Three-Quarters of Huntington's Disease Patients

Among dominant neurodegenerative disorders, Huntingtons disease (HD) is perhaps the best candidate for treatment with small interfering RNAs (siRNAs) [1-9]. Invariably fatal, HD is caused by expansion of a CAG repeat in the Huntingtin gene, creating an extended polyglutamine tract that makes the Huntingtin protein toxic [10]. Silencing mutant Huntingtin messenger RNA (mRNA) should provide therapeutic benefit, but normal Huntingtin likely contributes to neuronal function [11-13]. No siRNA strategy can yet distinguish among the normal and disease Huntingtin alleles and other mRNAs containing CAG repeats [14]. siRNAs targeting the disease isoform of a heterozygous single-nucleotide polymorphism (SNP) in Huntingtin provide an alternative [15-19]. We sequenced 22 predicted SNP sites in 225 human samples corresponding to HD and control subjects. We find that 48% of our patient population is heterozygous at a single SNP site; one isoform of this SNP is associated with HD. Several other SNP sites are frequently heterozygous. Consequently, five allele-specific siRNAs, corresponding to just three SNP sites, could be used to treat three-quarters of the United States and European HD patient populations. We have designed and validated selective siRNAs for the three SNP sites, laying the foundation for allele-specific RNA interference (RNAi) therapy for HD.