Altered localization and functionality of TAR DNA Binding Protein 43 (TDP-43) in niemann- pick disease type C.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by the occurrence of visceral and neurological symptoms. At present, the molecular mechanisms causing neurodegeneration in this disease are unknown. Here we report the altered expression and/or mislocalization of the TAR-DNA binding protein 43 (TDP-43) in both NPC mouse and in a human neuronal model of the disease. We also report the neuropathologic study of a NPC patient's brain, showing that while TDP-43 is below immunohistochemical detection in nuclei of cerebellar Purkinje cells, it has a predominant localization in the cytoplasm of these cells. From a functional point of view, the TDP-43 mislocalization, that occurs in a human experimental neuronal model system, is associated with specific alterations in TDP-43 controlled genes. Most interestingly, treatment with N-Acetyl-cysteine (NAC) or beta-cyclodextrin (CD) can partially restore TDP-43 nuclear localization. Taken together, the results of these studies extend the role of TDP-43 beyond the Amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD)/Alzheimer disease (AD) spectrum. These findings may open novel research/therapeutic avenues for a better understanding of both NPC disease and the TDP-43 proteinopathy disease mechanism.

TCL1 transgenic mouse model as a tool for the study of therapeutic targets and microenvironment in human B-cell chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy with a mature phenotype. In spite of its relatively indolent nature, no radical cure is as yet available. CLL is not associated with either a unique cytogenetic or a molecular defect, which might have been a potential therapeutic target. Instead, several factors are involved in disease development, such as environmental signals which interact with genetic abnormalities to promote survival, proliferation and an immune surveillance escape. Among these, PI3-Kinase signal pathway alterations are nowadays considered to be clearly important. The TCL1 gene, an AKT co-activator, is the cause of a mature T-cell leukemia, as well as being highly expressed in all B-CLL. A TCL1 transgenic mouse which reproduces leukemia with a distinct immunophenotype and similar to the course of the human B-CLL was developed several years ago and is widely used by many groups. This is a review of the CLL biology arising from work of many independent investigators who have used TCL1 transgenic mouse model focusing on pathogenetic, microenviroment and therapeutic targets.

A marked paucity of granule cells in the developing cerebellum of the Npc1(-/-) mouse is corrected by a single injection of hydroxypropyl-ß-cyclodextrin.

In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1(-/-) mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%-25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent. Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8-PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1(-/-) mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1(-/-) mice. A single injection of hydroxypropyl-ß-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size. To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum.

Cholesterol metabolism-associated molecules in late onset Alzheimer's disease.

Alzheimer’s disease (AD) is the most common cause of dementia and, with an aging population, poses a huge public health problem. Although a small per cent is caused by single gene changes, most AD is sporadic and unexplained. Of many modifying factors, changes in brain cholesterol homeostasis are the best studied. We present a review of the role of altered cholesterol metabolism and hypercholesterolemia in APP processing and Abeta generation. We also provide an overview of the potential pharmacological modulation of cholesterol homeostasis in the brain by cholesterol-lowering agents and beta-cyclodextrins.

Strain-specific proportion of the two isoforms of the dopamine D2 receptor in the mouse striatum: associated neural and behavioral phenotypes.

Genetic variability in the proportion of the two alternative dopamine D2 receptor (D2R) mRNA splice variants, D2R-long (D2L) and D2R-short (D2S), influence corticostriatal functioning and could be implicated in liability to psychopathology. This study compared mesostriatal D2L/D2S ratios and associated neural and behavioral phenotypes in mice of the DBA/2J and C57BL/6J-inbred strains, which differ for schizophrenia- and addiction-like phenotypes. Results showed that DBA/2J mice lack the striatal predominance of D2L that has been reported in the rat and in C57BL/6J mice and confirmed in the latter strain by this study. Only C57BL/6J mice showed enhanced striatal c-Fos expression under D1R and D2/3R co-stimulation, indicating synergistic interaction between the subtypes of DA receptors. Instead, DBA/2J mice were characterized by opposing effects of D2/3R and D1R stimulation on striatal c-Fos expression, in line with a more pronounced influence of D2S isoform, and did not express stereotyped climbing under D1R and D2/3R co-stimulation, as reported for D2L-/- mice. Finally, strain-specific modulation of c-Fos expression by D1R and D2/3R co-stimulation was selectively observed in striatal compartments receiving inputs from the prefrontal cortex and involved in the control of motivated behaviors. These results show differences in tissue-specific D2R splicing in mice with intact genotypes and support a role for this phenotype in individual variability of corticostriatal functioning and in liability to psychopathology.

Expression screening for Lhx3 downstream genes identifies Thg-1pit as a novel mouse gene involved in pituitary development.

Thg-1pit, a novel mouse gene, was detected in a screen for genes that are differentially expressed in the developing pituitary of wild-type and Lhx3 null mutant embryos. The predicted translation product of the Thg-1pit gene contains a C-terminal TSC-box adjacent to a leucine zipper motif. These features are characteristic for the TSC-22/DIP/bun family of proteins. The onset of prominent Thg-1pit expression coincides with Lhx3 activation at early stages of pituitary development. Expression is further enhanced as cells begin to differentiate within the developing pituitary gland. No expression is observed in the pituitary rudiment of mutants that lack Lhx3 function. A possible role is thus suggested for Lhx3 activities in the regulation of Thg-1pit function during early steps of pituitary organogenesis.

Growing dictyate oocytes, but not early preimplantation embryos, of the mouse display high levels of DNA homologous recombination by single-strand annealing and lack DNA nonhomologous end joining.

We have investigated the ability of growing dictyate oocytes and early preimplantation embryos of the mouse to process extrachromosomal DNA molecules with free ends by intranuclearly microinjecting DNA fragments containing a region of homology of various extent at either the 5' or 3' terminus. Homologous recombination of these fragments by single-strand annealing (SSA), but not other DNA recombination/joining mechanisms, resulted in the formation of a full-length hsp-lacZ-pA fusion gene that was transcriptionally activated by heat shock in growing oocytes and spontaneously at the early two-cell stage in the embryos, making it possible to quantitatively evaluate SSA activities of these cells by the beta-galactosidase produced. SSA activities of oocytes and embryos were similar in their general properties and in the activity levels observed with saturating amounts of DNA. However, embryo SSA was almost one order of magnitude less effective than that of oocytes. Oocyte and embryo 5' --> 3' exonuclease (a key function of the SSA pathway) and DNA nonhomologous end joining (NHEJ) activities were also investigated using an asymmetric PCR assay. Results showed that NHEJ is lacking in oocytes and is very prominent in the embryos, where it competes with SSA for the injected DNA.

A developmentally regulated GAGA box-binding factor and Sp1 are required for transcription of the hsp70.1 gene at the onset of mouse zygotic genome activation.

We have investigated the onset of zygotic genome transcription in early two-cell mouse embryos by analyzing the regulation of hsp70.1, one of the first genes expressed after fertilization. The transcriptional activation of both an episomic hsp70 promoter and the endogenous hsp70.1 gene requires the contiguity of the GC box proximal to the TATA box with a GAGA box and involves GC box- and GAGA box-binding factors. In vivo transcription factor titrations with double-stranded oligodeoxyribonucleotides and antibodies pinpoint these factors as Sp1 and a novel murine GAGA box-binding factor, which is structurally related to the Drosophila GAGA factor and acts as transcriptional coactivator/potentiator of Sp1. Mouse unfertilized eggs and one-cell and two-cell embryos display a GAGA box-binding activity of maternal origin that disappears at the four-cell stage and is also abundant in the gonads, but is barely detectable in other adult tissues. In light of the well-established nucleosome-disruption role of the Drosophila GAGA factor, these findings suggest a novel mechanism of enhancer-independent gene derepression in early mouse embryos.

Quantitative RT-PCR amplification of RNA in single mouse oocytes and preimplantation embryos.

We describe a simple whole-cell method for quantitative reverse transcription (RT) PCR amplification of RNA that consistently allows the analysis of trace amounts of RNA, such as those carried by a fraction of a single mouse oocyte or preimplantation embryo, without organic extraction. The method is based on a preliminary genomic DNA digestion by DNase I in the presence of Mn++ and a subsequent RT step with rTth Reverse Transcriptase at 70 degrees C with the same buffer components, which also has the effect to irreversibly denature DNase I activity. Because of the completeness of genomic DNA digestion and RNA recovery, this procedure makes it possible to quantitatively amplify any target RNA, including those coded by intronless genes or genes whose intron-exon boundaries are unknown. By taking mRNAs of beta-actin, heat-shock protein HSP70.1 and ribosomal protein S16 as experimental models, we demonstrate the effectiveness of genomic DNA digestion by DNase I-Mn++ and of DNase I heat-denaturation and the quantitative properties of our method. We also show that this procedure is useful for transcriptional analyses during development that are hindered by paucity of biological material.

Developmental activation of an episomic hsp70 gene promoter in two-cell mouse embryos by transcription factor Sp1.

To investigate the control of zygotic genome expression in two-cell mouse embryos, we studied transcription factors required for transient expression of microinjected DNA constructs driven by the promoter of one of the earliest genes activated after fertilization in this system, the heat shock gene hsp70. Cis-acting elements required for hsp70 activation were first investigated by mutational analysis. Mutation of the TATA box and a proximal GC box strongly inhibited construct expression, while that of a CCAAT box had no effect. Transcription factors binding the wild-type hsp70 promoter were then titrated in vivo by coinjecting the construct with double-stranded oligodeoxyribonucleotides containing definite consensus sequences. Wild-type GC box oligonucleotides strongly inhibited construct expression, while those containing mutated GC boxes, wild-type CCAAT boxes, and heat shock elements had no effects. Finally, construct expression was challenged by coinjecting antibodies to specific transcription factors. Antibodies to factor Sp1 depressed construct expression in a dose-dependent manner, while those to Sp2, HSF1 and HSF2 were ineffective. These results pinpoint the Sp1 transcription factor as an absolute requirement for activation of the hsp70 gene promoter in two-cell mouse embryos, and make this factor a candidate for a major regulator of the onset of murine zygotic genome expression.

Complex expression of murine heat shock transcription factors.

A central step in the transcriptional activation of heat shock genes is the binding of the heat shock factor (HSF) to upstream heat shock elements (HSEs). In vertebrates, HSF1 mediates the ubiquitous response to stress stimuli, while the role of a second HSE-binding factor, HSF2, is still unclear. In this work we show that both factors are expressed in a wide range of murine tissues and each exists as two splicing isoforms. Although HSFs are virtually ubiquitous proteins, their abundance is predominant in testis and variable among other tissues, indicating specific regulations of their expression. A low level of DNA-binding activity of HSF1, detected in many tissues, is probably physiological and is not explained by an anomalous regulation of one of the two isoforms. Our observations suggest that these regulatory proteins may all have roles in fully developed tissues. This possibility is not mutually exclusive of a role of HSF2 during cellular differentiation and tissue development [L. Sistonen, K. D. Sarge and R. I. Morimoto (1994), Mol. Cell. Biol., 14, 2087-2099].

Hyperthermia specifically inhibits bivalent chromosome disjunction in maturing mouse oocytes.

The effect of hyperthermia on mammalian oocyte maturation was studied by allowing preovulatory mouse oocytes to mature spontaneously for 17 h in vitro under controlled temperature conditions. At the end of culture, oocytes were screened for their maturation stage and for chromosome morphology and number. Mild hyperthermic conditions (38.5-40.0 degrees C) during maturation specifically disturbed the process of bivalent chromosome disjunction, but not other maturation steps, by blocking oocytes at the metaphase I stage and preventing cells from entering subsequent maturation steps. Some oocytes that had reached metaphase II under hyperthermic conditions had chromosome imbalance. Oocytes matured at 40.0 degrees C displayed chromosome morphological abnormalities, including altered sister chromatid separation and nucleus/nuclei formation, at a frequency significantly higher than oocytes matured at 37.0-39.0 degrees C. When incubation temperature was raised above 40.0 degrees C, increasing fractions of oocytes were inhibited from entering initial maturation steps. We conclude that hyperthermia during mammalian oocyte maturation specifically damages the process of bivalent chromosome disjunction and induces the appearance of chromosome structural defects and imbalance in unfertilized eggs.

Developmental regulation of heat-shock response in mouse oogenesis: identification of differentially responsive oocyte classes during Graafian follicle development.

The response to heat (hs response) of dictyate mouse oocytes at various differentiation stages was analyzed in vitro, by determining patterns of oocyte heat-shock (hs) gene expression and heat-shock protein (HSP) synthesis, under both normal conditions and after an hs. Growing oocytes constitutively synthesized HSP89 and HSC70, and, in contrast to preovulatory oocytes which do not display an hs response, displayed a heat-elicited, transcription-dependent synthesis of two HSP68 isoforms, but not of other inducible HSPs. To determine the developmental schedule of hs response disappearance during oogenesis, fully grown oocytes from Graafian follicles were morphologically sorted into three discrete classes related to the follicle development, namely, loosely associated with granulosa cells (LA oocytes, from small Graafian follicles), intermediately associated with granulosa cells (IA oocytes, from medium-sized Graafian follicles), and cumulus-associated (CA oocytes, from mature follicles). LA oocytes displayed an hs response qualitatively similar to, but smaller in extent than, that of growing oocytes, and were able to resume and complete spontaneous meiotic maturation in vitro at a high rate after hs. We conclude that hs response of mouse dictyate oocytes is maximal during growth period, significantly declines with acquisition of full oocyte size and antrum formation within the follicle, and is finally shut off with oocyte/follicle terminal differentiation.