Eric K. Richfield

EIF2B5 mutations compromise GFAP+ astrocyte generation in vanishing white matter leukodystrophy.

Vanishing white matter disease (VWM) is a heritable leukodystrophy linked to mutations in translation initiation factor 2B (eIF2B). Although the clinical course of this disease has been relatively well described, the cellular consequences of EIF2B mutations on neural cells are unknown. Here we have established cell cultures from the brain of an individual with VWM carrying mutations in subunit 5 of eIF2B (encoded by EIF2B5). Despite the extensive demyelination apparent in this VWM patient, normal-appearing oligodendrocytes were readily generated in vitro. In contrast, few GFAP-expressing (GFAP+) astrocytes were present in primary cultures, induction of astrocytes was severely compromised, and the few astrocytes generated showed abnormal morphologies and antigenic phenotypes. Lesions in vivo also lacked GFAP+ astrocytes. RNAi targeting of EIF2B5 severely compromised the induction of GFAP+ cells from normal human glial progenitors. This raises the possibility that a deficiency in astrocyte function may contribute to the loss of white matter in VWM leukodystrophy.

Targeted disruption of Zfp36l2, encoding a CCCH tandem zinc finger RNA-binding protein, results in defective hematopoiesis

Members of the tristetraprolin family of tandem CCCH finger proteins can bind to AU-rich elements in the 3-untranslated region of mRNAs, leading to their deadenylation and subsequent degradation. Partial deficiency of 1 of the 4 mouse tristetraprolin family members, Zfp36l2, resulted in complete female infertility because of early embryo death. We have now generated mice completely deficient in the ZFP36L2 protein. Homozygous Zfp36l2 knockout (KO) mice died within approximately 2 weeks of birth, apparently from intestinal or other hemorrhage. Analysis of peripheral blood from KO mice showed a decrease in red and white cells, hemoglobin, hematocrit, and platelets. Yolk sacs from embryonic day 11.5 (E11.5) Zfp36l2 KO mice and fetal livers from E14.5 KO mice gave rise to markedly reduced numbers of definitive multilineage and lineage-committed hematopoietic progenitors. Competitive reconstitution experiments demonstrated that Zfp36l2 KO fetal liver hematopoietic stem cells were unable to adequately reconstitute the hematopoietic system of lethally irradiated recipients. These data establish Zfp36l2 as a critical modulator of definitive hematopoiesis and suggest a novel regulatory pathway involving control of mRNA stability in the life cycle of hematopoietic stem and progenitor cells.

Zfp36l3, a Rodent X Chromosome Gene Encoding a Placenta-Specific Member of the Tristetraprolin Family of CCCH Tandem Zinc Finger Proteins

Abstract Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger (TZF) proteins can bind directly to AU-rich elements (ARE) in mRNA, causing deadenylation and destabilization of the transcripts to which they bind. We describe here a novel fourth mammalian member of the TTP protein family, designated ZFP36L3, which could also bind directly to ARE-containing RNAs and could promote the deadenylation and degradation of ARE-containing target RNAs. Zfp36l3 transcript expression was detected only in placenta and extraembryonic tissues in the mouse. It was expressed throughout development in the placenta and was particularly highly expressed in the cells of the labyrinthine layer of the trophoblastic placenta. Unlike the other family members, the expression of a ZFP36L3-green fluorescent protein fusion protein was entirely cytoplasmic when expressed in 293 cells, even in the presence of the CRM1-dependent nuclear export inhibitor leptomycin B. Zfp36l3 was located on the mouse X chromosome; a similar predicted gene was present on the rat X chromosome, but there was no evidence for a similar gene in humans. ZFP36L3 may thus be a rodent-specific or even murine-specific member of the TTP protein family. Its presumed role in placental physiology may be unique to rodents or murine rodents, but this role may be subsumed by other family members in nonrodents.

Toxicokinetics and toxicodynamics of paraquat accumulation in mouse brain

Paraquat (PQ) is a potential human neurotoxicant and is used in models of oxidative stress. We determined the toxicokinetics (TK) and toxicodynamics (TD) of PQ in adult mouse brain following repeated or prolonged PQ exposure. PQ accumulated in different brain regions and reached a plateau after approximately 18 i.p. (10 mg/kg) doses and resulted in modest morbidity and mortality unpredictably associated with dose interval and number. PQ had divergent effects on horizontal locomotor behavior depending on the number of doses. PQ decreased striatal dopamine levels after the 18th to 36th i.p. dose (10 mg/kg) and reduced the striatal level of tyrosine hydroxylase. Drinking water exposure to PQ (0.03- 0.05 mg/ml) did not result in any mortality and resulted in concentration and time dependent levels in the brain. The brain half-life of PQ varied with mouse strain. PQ accumulates and may saturate a site in mouse brain resulting in complex PQ level and duration-related consequences. These findings should alter our risk assessment of this compound and demonstrate a useful, but complex dynamic model for understanding the consequences of PQ in the brain.

Genetic-based, differential susceptibility to paraquat neurotoxicity in mice

Paraquat (PQ) is an herbicide used extensively in agriculture. This agent is also suspected to be a risk factor for Parkinsons disease (PD) by harming nigro-striatal dopamine neurons. There is likely, genetic-based, individual variability in susceptibility to PQ neurotoxicity related PD. In this study, we measured the delivery of PQ to the brain after three weekly injections of PQ at 5 mg kg(-1), PQ-related neural toxicity after three weekly injections of PQ at 1 mg kg(-1)or 5 mg kg(-1), PQ-related iron accumulation and PQ-related gene expression in midbrain of DBA/2J (D2) and C57BL/6J (B6) inbred mouse strains after a single injection of PQ at 15 mg kg(-1) and 10 mg kg(-1), respectively. Results showed that compared to controls, PQ-treated B6 mice lost greater numbers of dopaminergic neurons in the substantia nigra pars compacta than D2 mice; however, distribution of PQ to the midbrain was equal between the strains. PQ also significantly increased iron concentration in the midbrain of B6 but not D2 mice. Microarray analysis of the ventral midbrain showed greater PQ-induced changes in gene expression in B6 compared to D2 mice. This is the first study to report genetically-based differences in susceptibility to PQ neurotoxicity and to understanding individual differences in vulnerability to PQ neurotoxicity and its relation to PD in humans.

Quantification of Paraquat, MPTP, and MPP+ in brain tissue using microwave-assisted solvent extraction (MASE) and high-performance liquid chromatography–mass spectrometry

Animal models, consistent with the hypothesis of direct interaction of paraquat (PQ) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) with specific areas of the central nervous system have been developed to study Parkinson’s disease (PD) in mice. These models have necessitated the creation of an analytical method for unambiguous identification and quantitation of PQ and structurally similar MPTP and 1-methyl-4-phenylpyridinium ion (MPP+) in brain tissue. A method for determination of these compounds was developed using microwave-assisted solvent extraction (MASE) and liquid chromatography–mass spectrometry. Extraction solvent and microwave conditions such as power and time were optimized to produce recoveries of 90% for PQ 78% for MPTP and 97% for its metabolite MPP+. The chromatographic separation was performed on a C8, column and detection was carried out using an ion trap as an analyzer with electrospray ionization. Mass spectrometer parameters such as heated capillary temperature, spray voltage, capillary voltage and others were also optimized for each analyte. Analysis was done in selective ion-monitoring (SIM) mode using m/z 186 for PQ, m/z 174 for MPTP, and m/z 170 for MPP+. The method detection limit for paraquat in matrix was 100xa0pg, 40xa0pg for MPTP, and 20xa0pg MPP+.

Critical Role of Truncated α-Synuclein and Aggregates in Parkinson's Disease and Incidental Lewy Body Disease

The role of Lewy bodies, Lewy neurites and α‐synuclein (αSYN) in the pathophysiology and diagnosis of Parkinsons disease (PD) is unclear. We used postmortem human tissue, a panel of antibodies (Abs) and confocal microscopy to examine the three‐dimensional neurochemical anatomy of the nigrostriatal system. Abs were specific to truncated (tαSYN), phosphorylated and full‐length αSYN. The findings demonstrate the critical role of tαSYN in initiating aggregation, a role for other forms of αSYN in aggregate expansion, a reason for the wide variety of proteins present in different aggregates, an explanation for the laminar appearance of aggregates described historically using different methods, the existence of proximal greater than distal aggregation in the vulnerable nigrostriatal pathway, the independent transport of different forms of αSYN as cargo along axons and a possible sequence for the formation of Lewy bodies. Findings differed between incidental Lewy body disease and PD only quantitatively. These findings have implications for understanding the pathogenesis and treatment of PD.

Biochemical and morphological consequences of human α‐synuclein expression in a mouse α‐synuclein null background

A consensus about the functions of human wild‐type or mutated α‐synuclein (αSYN) is lacking. Both forms of αSYN are implicated in Parkinson’s disease, whereas the wild‐type form is implicated in substance abuse. Interactions with other cellular proteins and organelles may meditate its functions. We developed a series of congenic mouse lines containing various allele doses or combinations of the human wild‐type αSYN (hwαSYN) or a doubly mutated (A30P*A53T) αSYN (hm2αSYN) in a C57Bl/6J line spontaneously deleted in mouse αSYN (C57BL/6JOla). Both transgenes had a functional role in the nigrostriatal system, demonstrated by significant elevations in striatal catecholamines, metabolites and the enzyme tyrosine hydroxylase compared with null‐mice without a transgene. Consequences occurred when the transgenes were expressed at a fraction of the endogenous level. Hemizygous congenic mice did not exhibit any change in the number or size of dopaminergic neurons in the ventral midbrain at 9u2003months of age. Human αSYN was predominantly located in neuronal cell bodies, neurites, synapses, and in intraneuronal/intraneuritic aggregates. The hm2αSYN transgene resulted in more aggregates and dystrophic neurites than did the hw5 transgene. The hwαSYN transgene resulted in higher expression of two striatal proteins, synaptogamin 7 and UCHL1, compared with the levels of the hm2αSYN transgene. These observations suggest that mutations in αSYN may impair specific functional domains, leaving others intact. These lines may be useful for exploring interactions between hαSYN and environmental or genetic risk factors in dopamine‐related disorders using a mouse model.

Sporadic midbrain dopamine neuron abnormalities in laboratory mice

We report an anatomical abnormality of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) in different strains of inbred and outbred mice, one mouse strain (C57BL/6, B6) from different commercial suppliers, and in B6J mice bred internally. The abnormality consisted of a sporadic and unpredictable decrease in the number of dopaminergic neurons and/or a reduction or complete loss of tyrosine hydroxylase (TH) staining in a focal subset of neurons of the SNpc and/or VTA. This abnormality had a preference for a unilateral right side location, but could affect one or both sides of each subregion independently or together. The frequency and severity were variable between and within strains and colonies. The neuronal abnormality was found in mice from the five commercial suppliers examined, 5/15 inbred strains from a single supplier, and the one outbred strain (CD1) examined. The striatal content of catecholamines was not affected by this abnormality even when there was significant asymmetric TH neuronal loss, but did vary significantly between commercial suppliers. Manipulations in housing conditions did not affect the abnormalities. The mechanism and cause of this abnormality could not be determined in this study although several potential factors were eliminated. The frequent, but not universal, occurrence of this abnormality has significant implications for the use of laboratory mice in studying the midbrain dopamine system and warrants its recognition, knowledge of their frequency, and exploration of a mechanism to address or eliminate them.

259. Targeted Gene Silencing by U1 Adaptor Oligonucleotides in Preclinical Models of Parkinson's Disease and Huntington's Disease

Many candidate genes are implicated in neurodegenerative disease, but to study potential therapeutic effects of modifying their expression in the central nervous system of animal models has been difficult, often requiring slow, expensive transgenic methods. Transient gene silencing with synthetic oligonucleotides can be a fast, inexpensive alternative to making new transgenic animal models, and a complimentary technique to extend the utility of existing ones. For genes with products that have been validated as therapeutic targets, but are not amenable to small molecule drugs, gene silencing may also be the therapeutic modality of choice.U1 Adaptors are a third generation of oligonucleotide-mediated gene silencing technology, mechanistically distinct from antisense or siRNA. U1 Adaptors act by selectively interfering with a key step in mRNA maturation: the addition of a 3’ polyadenosine (polyA) tail. Nearly all protein-coding mRNAs require a polyA tail, and failure to add one results in rapid degradation of the nascent mRNA inside the nucleus, preventing expression of a protein product. U1 Adaptor oligonucleotides are well suited to in vivo applications because they can accept extensive chemical modifications to improve nuclease resistance and the attachment of bulky groups, such as tags for imaging or ligands for receptor-mediated uptake by target cells, without loss of silencing activity.To explore the feasibility of U1 Adaptor technology for CNS targets, we designed panels of candidate U1 Adaptor oligos for mouse Scna (alpha-synuclein) and human HTT (Huntingtin), and screened them in cell culture. We identified U1 Adaptors that robustly suppress mouse Scna mRNA and reduce alpha-synuclein protein levels in mouse cells. Similarly, we identified U1 Adaptors that suppress the predominant, full length human HTT mRNA and reduce HTT protein levels in human cells. We also identified U1 Adaptors that suppress the HTT exon-1 truncation isoform recently implicated in HD pathogenesis. For in-vivo PK/PD studies, U1 Adaptors were delivered into the CNS of mice, by intracerebroventricular (ICV) injection or by direct stereotaxic injections into the striatum. To examine distribution, cellular uptake and persistence over time, fluorescently tagged U1 Adaptors were administered, then visualized by confocal microscopy in brain sections. ICV injection achieved broad distribution of fluorescent U1 Adaptors throughout the brain, with uptake visible in most cells. Subcellular distribution 24 hours after injection was diffuse in both cytoplasmic and nuclear compartments, but became more punctate and perinuclear by 48 hours. U1 Adaptor oligonucleotides were detected on northern blots of small RNA recovered from brain tissue specimens. Their levels in tissue were estimated by comparison to a standard loading curve, and correlated well with ICV-injected dose. After direct stereotaxic injection to the striatum, U1 Adaptors diffused rapidly and widely, and were taken up by all striatial cells, though preferentially by neurons. Adaptors persisted in tissue for at least five days (the last time point assayed) and reached the nuclei of striatial cells. We then did a series of studies with U1 Adaptors specific for mouse Scna mRNA. RNAScope was used to visualize relative levels of mRNA in situ. Injection of U1 Adaptors directly into the striatum resulted in clearly reduced expression of Scna mRNA and also of mRNA for synaptophysin, known to be down-regulated when α-synuclein expression is reduced.