Duong P. Huynh

Nuclear localization or inclusion body formation of ataxin-2 are not necessary for SCA2 pathogenesis in mouse or human

Instability of CAG DNA trinucleotide repeats is the mutational mechanism for several neurodegenerative diseases resulting in the expansion of a polyglutamine (polyQ) tract. Proteins with long polyQ tracts have an increased tendency to aggregate, often as truncated fragments forming ubiquitinated intranuclear inclusion bodies. We examined whether similar features define spinocerebellar ataxia type 2 (SCA2) pathogenesis using cultured cells, human brains and transgenic mouse lines. In SCA2 brains, we found cytoplasmic, but not nuclear, microaggregates. Mice expressing ataxin-2 with Q58 showed progressive functional deficits accompanied by loss of the Purkinje cell dendritic arbor and finally loss of Purkinje cells. Despite similar functional deficits and anatomical changes observed in ataxin-1[Q80] transgenic lines, ataxin-2[Q58] remained cytoplasmic without detectable ubiquitination.

Learning deficits, but normal development and tumor predisposition, in mice lacking exon 23a of Nf1

Neurofibromatosis type 1 (NF1) is a commonly inherited autosomal dominant disorder. Previous studies indicated that mice homozygous for a null mutation in Nf1 exhibit mid-gestation lethality, whereas heterozygous mice have an increased predisposition to tumors and learning impairments. Here we show that mice lacking the alternatively spliced exon 23a, which modifies the GTPase-activating protein (GAP) domain of Nf1, are viable and physically normal, and do not have an increased tumor predisposition, but show specific learning impairments. Our findings have implications for the development of a treatment for the learning disabilities associated with NF1 and indicate that the GAP domain of NF1 modulates learning and memory.

Expression of ataxin-2 in brains from normal individuals and patients with Alzheimer's disease and spinocerebellar ataxia 2

Spinocerebellar ataxia type 2 (SCA2) is caused by expansion of a CAG trinucleotide repeat located in the coding region of the human SCA2 gene. The SCA2 gene product, ataxin‐2, is a basic protein with two domains (Sm1 and Sm2) implicated in RNA splicing and protein interaction. However, the wild‐type function of ataxin‐2 is yet to be determined. To help clarify the function of ataxin‐2, we produced antibodies to three antigenic peptides of ataxin‐2 and analyzed the expression pattern of ataxin‐2 in normal and SCA2 adult brains and cerebellum at different developmental stages. These studies revealed that (1) both wild‐type and mutant forms of ataxin‐2 were synthesized; (2) the wild‐type ataxin‐2 was localized in the cytoplasm in specific neuronal groups with strong labeling of Purkinje cells; (3) the level of ataxin‐2 increased with age in Purkinje cells of normal individuals; and (4) ataxin‐2‐like immunoreactivity in SCA2 brain tissues was more intense than in normal brain tissues, and intranuclear ubiquitinated inclusions were not seen in SCA2 brain tissues. Ann Neurol 1999;45:232–241

Parkin is associated with actin filaments in neuronal and nonneural cells

Inactivating mutations of the gene encoding parkin are responsible for autosomal recessive juvenile parkinsonism (AR‐JP). However, little information is known about the function and distribution of parkin. We generated antibodies to two different peptides of parkin. By Western blot analysis and immunohistochemistry, we found that parkin is a 50‐kd protein that is expressed in neuronal processes and cytoplasm of selected neurons in the basal ganglia, midbrain, cerebellum, and cerebral cortex. Unlike ubiquitin and α‐synuclein, parkin labeling was not found in Lewy bodies of four sporadic Parkinson disease brains. Parkin was colocalized with actin filaments but not with microtubules in COS1 kidney cells and nerve growth factor–induced PC12 neurons. These results point to the importance of the cytoskeleton and associated proteins in neurodegeneration. Ann Neurol 2000;48:737–744

Immunohistochemical detection of schwannomin and neurofibromin in vestibular schwannomas, ependymomas and meningiomas

In addition to schwannomas, patients with neurofibromatosis type 2 (NF2) frequently develop meningiomas and occasionally, ependymomas. Using DNA and protein analyses, we have shown NF2 gene mutations and lack of the gene product schwannomin in 29 schwannomas, 10 meningiomas, and in 7 ependymomas. We have raised antibodies (ABs) to peptides from the C-terminal (5990-AB) and N-terminal (5991-AB) domains of schwannomin. The ABs specifically detected a 65 kDa protein in a Schwann cell line and recognized schwannomin in the cytoplasm of Schwann cells (SCH), perineurial cells, and vestibular ganglion neurons. None of the 29 schwannomas were stained by the 5990-AB. Only 4 schwannomas were stained by the 5991-AB, indicating that most truncated schwannomins were unstable or not expressed in schwannomas. Seven of 10 meningiomas, including 3 tumors from NF2 patients, were not stained by either 5990-AB or 5991-AB. Only 2 of 7 ependymomas lacked schwannomin. Complete lack of schwannomin in these tumors supports a tumor suppressor function for schwannomin in some meningiomas and ependymomas. All tumors showed staining with an antibody to a C-terminal peptide of neurofibromin, confirming that full-length neurofibromin is present in these vestibular schwannomas, meningiomas, and ependymomas. The presence of schwannomin in some meningiomas and in the majority of ependymomas indicates that additional genes are likely to play a role in tumorigenesis of these tumors.

Identification and expression of a mouse ortholog of A2BP1

Human ataxin-2 contains a polyglutamine repeat that is expanded in patients with spinocerebellar ataxia type 2 (SCA2). Ataxin-2 is highly conserved in evolution with orthologs in mouse, Caenorhabditis elegans, and Drosophila melanogaster. It interacts at its C-terminus with ataxin-2 binding protein 1, A2BP1. This study presents a highly conserved mouse ortholog of A2BP1, designated A2bp1. The amino acid sequence of the human and mouse protein is 97.6% identical. This remarkable degree of conservation supports the fact that these proteins have an important basic function in development and differentiation. Sequence analysis reveals the existence of RNA binding motifs. The A2bp1 transcript was found in various regions of the CNS including cerebellum, cerebral cortex, brain stem, and thalamus/hypothalamus. The A2bp1 protein was detected by immunocytochemistry in the CNS and connective tissue of the mouse embryo starting at stage E11, as well as in the heart at all stages. Mouse embryos showed varying expression of A2bp1 at all stages. Previous studies in other model systems had implicated the orthologs of ataxin-2 and A2BP1 in development. This study suggests a role for A2bp1 in embryogenesis as well as in the adult nervous system, possibly mediated by a function in RNA distribution or processing.

Parkin is an E3 ubiquitin-ligase for normal and mutant ataxin-2 and prevents ataxin-2-induced cell death.

Expansion of the polyQ repeat in ataxin-2 results in degeneration of Purkinje neurons and other neuronal groups including the substantia nigra in patients with spinocerebellar ataxia type 2 (SCA2). In animal and cell models, overexpression of mutant ataxin-2 induces cell dysfunction and death, but little is known about steady-state levels of normal and mutant ataxin-2 and cellular mechanisms regulating their abundance. Based on preliminary findings that ataxin-2 interacted with parkin, an E3 ubiquitin ligase mutated in an autosomal recessive form of Parkinsonism, we sought to determine whether parkin played a role in regulating the steady-state levels of ataxin-2. Parkin interacted with the N-terminal half of normal and mutant ataxin-2, and ubiquitinated the full-length form of both wild-type and mutant ataxin-2. Parkin also regulated the steady-state levels of endogenous ataxin-2 in PC12 cells with regulatable parkin expression. Parkin reduced abnormalities in Golgi morphology induced by mutant ataxin-2 and decreased ataxin-2 induced cytotoxicity. In brains of SCA2 patients, parkin labeled cytoplasmic ataxin-2 aggregates in Purkinje neurons. These studies suggest a role for parkin in regulating the intracellular levels of both wild-type and mutant ataxin-2, and in rescuing cells from ataxin-2-induced cytotoxicity. The role of parkin variants in modifying the SCA2 phenotype and its use as a therapeutic target should be further investigated.

Dissociated Fear and Spatial Learning in Mice with Deficiency of Ataxin-2

Mouse models with physiological and behavioral differences attributable to differential plasticity of hippocampal and amygdalar neuronal networks are rare. We previously generated ataxin-2 (Atxn2) knockout mice and demonstrated that these animals lacked obvious anatomical abnormalities of the CNS, but showed marked obesity and reduced fertility. We now report on behavioral changes as a consequence of Atxn2-deficiency. Atxn2-deficiency was associated with impaired long-term potentiation (LTP) in the amygdala, but normal LTP in the hippocampus. Intact hippocampal plasticity was associated behaviorally with normal Morris Water maze testing. Impaired amygdala plasticity was associated with reduced cued and contextual fear conditioning. Conditioned taste aversion, however, was normal. In addition, knockout mice showed decreased innate fear in several tests and motor hyperactivity in open cage testing. Our results suggest that Atxn2-deficiency results in a specific set of behavioral and cellular disturbances that include motor hyperactivity and abnormal fear-related behaviors, but intact hippocampal function. This animal model may be useful for the study of anxiety disorders and should encourage studies of anxiety in patients with spinocerebellar ataxia type 2 (SCA2).

Neuronal Expression and Intracellular Localization of Presenilins in Normal and Alzheimer Disease Brains

The expression patterns of presenilin 1 (PS1) and presenilin 2 (PS2) in human normal and Alzheimer disease (AD) brains were investigated using antibodies to specific N-terminal peptides of PS1 (Alzh14A and Alzh14B) and PS2 (Alzh1A-AB). The antibodies to peptides Alzh14A (Alzh14A-AB) and Alzh14B (Alzh14B-AB) detected the full-length protein (∼63 kDa) and the N-terminal-processed fragment (36 kDa) of PS1, while the Alzh1A-AB detected mainly the N-terminal-processed fragment (36 kDa) of PS2. Immunofluorescent staining detected by confocal microscopy suggested that both native PS1 and PS2 are localized mainly in the Golgi/ER apparatus. Immunohistochemical studies of human temporal lobes from 2 normal and 5 sporadic Alzheimer brains revealed high levels of PS1 and PS2 expression in the granule cell layer and pyramidal neurons of the hippocampus. Strong immunoreactivity was found in reactive astrocytes and neurofibrillary tangles of all 5 Alzheimer brains. In contrast, only 2 sporadic Alzheimer brains showed presenilin-positive neuritic plaques. These observations suggest that presenilins may be involved in the pathology of some cases of sporadic AD.

Differential expression and tissue distribution of parkin isoforms during mouse development.

Mutations of the parkin gene are a cause of autosomal recessive juvenile parkinsonism. Although the parkin gene has been isolated from mouse, rat, and human, little is known about its expression in neural and nonneural tissues during development. In this study, we used a polyclonal antibody to a peptide downstream of the parkin ubiquitin domain to investigate (1) the differential expression of parkin isoforms in protein extracts from fetal and adult mouse tissues, and (2) the distribution of parkin in mouse fetal tissues at different developmental stages and in adult CNS tissues. By Western blot analyses, at least three isoforms of parkin of 22, 50, and 55 kDa were differentially expressed in mouse tissues. The p22 and p50 isoforms were found in fetal and adult mouse CNS tissues, while the p55 isoform was found only in adult tissues. The p50 isoform is the predominant form in both fetal and adult tissues. Immunolocalization in mouse fetuses showed that parkin was expressed only after neuronal differentiation. Although parkin was localized throughout the cytoplasm, the highest level of parkin was found in the neurites of both fetal and adult neurons.