Aldamaria Puliti

Purkinje neuron Ca2+ influx reduction rescues ataxia in SCA28 model

Spinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disease caused by mutations of the mitochondrial protease AFG3L2. The SCA28 mouse model, which is haploinsufficient for Afg3l2, exhibits a progressive decline in motor function and displays dark degeneration of Purkinje cells (PC-DCD) of mitochondrial origin. Here, we determined that mitochondria in cultured Afg3l2-deficient PCs ineffectively buffer evoked Ca²⁺ peaks, resulting in enhanced cytoplasmic Ca²⁺ concentrations, which subsequently triggers PC-DCD. This Ca²⁺-handling defect is the result of negative synergism between mitochondrial depolarization and altered organelle trafficking to PC dendrites in Afg3l2-mutant cells. In SCA28 mice, partial genetic silencing of the metabotropic glutamate receptor mGluR1 decreased Ca²⁺ influx in PCs and reversed the ataxic phenotype. Moreover, administration of the β-lactam antibiotic ceftriaxone, which promotes synaptic glutamate clearance, thereby reducing Ca²⁺ influx, improved ataxia-associated phenotypes in SCA28 mice when given either prior to or after symptom onset. Together, the results of this study indicate that ineffective mitochondrial Ca²⁺ handling in PCs underlies SCA28 pathogenesis and suggest that strategies that lower glutamate stimulation of PCs should be further explored as a potential treatment for SCA28 patients.

The HIV-1 Viral Protein Tat Increases Glutamate and Decreases GABA Exocytosis from Human and Mouse Neocortical Nerve Endings

Human immunodeficiency virus-1 (HIV-1)-encoded transactivator of transcription (Tat) potentiated the depolarization-evoked exocytosis of [(3)H]D-aspartate ([(3)H]D-ASP) from human neocortical terminals. The metabotropic glutamate (mGlu) 1 receptor antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) prevented this effect, whereas the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) was ineffective. Western blot analysis showed that human neocortex synaptosomes possess mGlu1 and mGlu5 receptors. Tat potentiated the K(+)-evoked release of [(3)H]D-ASP or of endogenous glutamate from mouse neocortical synaptosomes in a CPCCOEt-sensitive and MPEP-insensitive manner. Deletion of mGlu1 receptors (crv4/crv4 mice) or mGlu5 receptors (mGlu5(-/-)mouse) silenced Tat effects. Tat enhanced inositol 1,4,5-trisphosphate production in human and mouse neocortical synaptosomes, consistent with the involvement of group I mGlu receptors. Tat inhibited the K(+)-evoked release of [(3)H]gamma-aminobutyric acid ([(3)H]GABA) from human synaptosomes and that of endogenous GABA or [(3)H]GABA from mouse nerve terminals; the inhibition was insensitive to CPCCOEt or MPEP. Tat-induced effects were retained by Tat(37-72) but not by Tat(48-85). In mouse neocortical slices, Tat facilitated the K(+)- and the veratridine-induced release of [(3)H]D-ASP in a CPCCOEt-sensitive manner and was ineffective in crv4/crv4 mouse slices. These observations are relevant to the comprehension of the pathophysiological effects of Tat in central nervous system and may suggest new potential therapeutic approaches to the cure of HIV-1-associated dementia.

Albuminuria and glomerular damage in mice lacking the metabotropic glutamate receptor 1

The metabotropic glutamate (mGlu) receptor 1 (GRM1) has been shown to play an important role in neuronal cells by triggering, through calcium release from intracellular stores, various signaling pathways that finally modulate neuron excitability, synaptic plasticity, and mechanisms of feedback regulation of neurotransmitter release. Herein, we show that Grm1 is expressed in glomerular podocytes and that a glomerular phenotype is exhibited by Grm1(crv4) mice carrying a spontaneous recessive inactivating mutation of the gene. Homozygous Grm1(crv4/crv4) and, to a lesser extent, heterozygous mice show albuminuria, podocyte foot process effacement, and reduced levels of nephrin and other proteins known to contribute to the maintenance of podocyte cell structure. Overall, the present data extend the role of mGlu1 receptor to the glomerular filtration barrier. The regulatory action of mGlu1 receptor in dendritic spine morphology and in the control of glutamate release is well acknowledged in neuronal cells. Analogously, we speculate that mGlu1 receptor may regulate foot process morphology and intercellular signaling in the podocyte.

Sequence and characterisation of the RET proto-oncogene 5' flanking region: Analysis of retinoic acid responsiveness at the transcriptional level

The RET proto‐oncogene encodes a receptor tyrosine kinase expressed during neural crest development. RET expression is enhanced in vitro by several differentiating agents, including retinoic acid (RA), which up‐regulates RET expression in neuroblastoma cell lines. In the present work we sequenced and analysed a 5 kbp genomic fragment 5′ to RET. Three deletion fragments of this region were tested for their RA inducibility in transient transfection assays and failed to support the hypothesis of a direct transcriptional activation. Finally, our functional analysis of a candidate RA response element present in the RET promoter provides new hints for the understanding of the interaction between nuclear receptors and their specific recognition sites.

Nephrin expression in adult rodent central nervous system and its interaction with glutamate receptors.

Nephrin is an immunoglobulin‐like adhesion molecule first discovered as a major component of the podocyte slit diaphragm, where its integrity is essential to the function of the glomerular filtration barrier. Outside the kidney, nephrin has been shown in other restricted locations, most notably in the central nervous system (CNS) of embryonic and newborn rodents. With the aim of better characterizing nephrin expression and its role in the CNS of adult rodents, we studied its expression pattern and possible binding partners in CNS tissues and cultured neuronal cells and compared these data to those obtained in control renal tissues and podocyte cell cultures. Our results show that, besides a number of locations already found in embryos and newborns, endogenous nephrin in adult rodent CNS extends to the pons and corpus callosum and is expressed by granule cells and Purkinje cells of the cerebellum, with a characteristic alternating expression pattern. In primary neuronal cells we find nephrin expression close to synaptic proteins and demonstrate that nephrin co‐immunoprecipitates with Fyn kinase, glutamate receptors and the scaffolding molecule PSD95, an assembly that is reminiscent of those made by synaptic adhesion molecules. This role seems to be confirmed by our findings of impaired maturation and reduced glutamate exocytosis occurring in Neuro2A cells upon nephrin silencing. Of note, we disclose that the very same nephrin interactions occur in renal glomeruli and cultured podocytes, supporting our hypothesis that podocytes organize and use similar molecular intercellular signalling modules to those used by neuronal cells. Copyright

De novo deletion of chromosome 11q12.3 in monozygotic twins affected by Poland Syndrome

BackgroundPoland Syndrome (PS) is a rare disorder characterized by hypoplasia/aplasia of the pectoralis major muscle, variably associated with thoracic and upper limb anomalies. Familial recurrence has been reported indicating that PS could have a genetic basis, though the genetic mechanisms underlying PS development are still unknown.Case presentationHere we describe a couple of monozygotic (MZ) twin girls, both presenting with Poland Syndrome. They carry a de novo heterozygous 126 Kbp deletion at chromosome 11q12.3 involving 5 genes, four of which, namely HRASLS5, RARRES3, HRASLS2, and PLA2G16, encode proteins that regulate cellular growth, differentiation, and apoptosis, mainly through Ras-mediated signaling pathways.ConclusionsPhenotype concordance between the monozygotic twin probands provides evidence supporting the genetic control of PS. As genes controlling cell growth and differentiation may be related to morphological defects originating during development, we postulate that the observed chromosome deletion could be causative of the phenotype observed in the twin girls and the deleted genes could play a role in PS development.

Knocking down metabotropic glutamate receptor 1 improves survival and disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease reflecting degeneration of upper and lower motoneurons (MNs). The cause of ALS and the mechanisms of neuronal death are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate (Glu)-mediated excitotoxicity plays a major role in MN degeneration in ALS. We recently demonstrated that the activation of Group I metabotropic Glu autoreceptors, belonging to both type 1 and type 5 receptors (mGluR1 and mGluR5), at glutamatergic spinal cord nerve terminals, produces excessive Glu release in mice over-expressing human superoxide-dismutase carrying the G93A point mutation (SOD1(G93A)), a widely used animal model of human ALS. To establish whether these receptors are implicated in ALS, we generated mice expressing half dosage of mGluR1 in the SOD1(G93A) background (SOD1(G93A)Grm1(crv4/+)), by crossing the SOD1(G93A) mutant mouse with the Grm1(crv4/+) mouse, lacking mGluR1 because of a spontaneous recessive mutation. SOD1(G93A)Grm1(crv4/+) mice showed prolonged survival probability, delayed pathology onset, slower disease progression and improved motor performances compared to SOD1(G93A) mice. These effects were associated to reduction of mGluR5 expression, enhanced number of MNs, decreased astrocyte and microglia activation, normalization of metallothionein and catalase mRNA expression, reduced mitochondrial damage, and decrease of abnormal Glu release in spinal cord of SOD1(G93A)Grm1(crv4/+)compared to SOD1(G93A) mice. These results demonstrate that a lower constitutive level of mGluR1 has a significant positive impact on mice with experimental ALS, thus providing the rationale for future pharmacological approaches to ALS by selectively blocking Group I metabotropic Glu receptors.

Deleted and normal chromosome 10 homologs from a patient with Hirschsprung disease isolated in two cell hybrids through enrichment by immunomagnetic selection.

A cytogenetically detectable deletion, del(10) (q11.2-->q21.2), was observed in a patient with total colonic aganglionosis with small bowel involvement (TCSA), a variant of Hirschsprung disease (HSCR). A similar deletion is present in another TCSA patient (S.M. Huson, personal communication). To reveal cytogenetically undetectable deletions of chromosome 10 in further patients, we developed a strategy for mapping chromosome 10 DNA markers with respect to the observed deletions. To this end, the two chromosome 10 homologs (deleted and normal) were segregated in two distinct somatic cell hybrids obtained after fusion of the patients fibroblasts with a Chinese hamster ovary cell line (YH21). Hybrid cells containing chromosome 10 were selected for the expression of the gene coding for the beta subunit of the fibronectin receptor (FNRB), which maps to 10p11.2, using a monoclonal antibody against FNRB. Hybrid 185.O contains the deleted chromosome, whereas hybrid 179.Q contains the nondeleted one. Southern blot and PCR analysis of DNA from these two hybrids mapped the markers RBP3H4, RET, D10S15, D10S5, D10S22, and D10S88 inside the deletion and D10S170, CDC2, EGR2, and D10S19 outside the deletion. MEN2A and MEN2B have recently been mapped within the centromeric region closely linked to RBP3 and D10S15 (which are located inside the deletion) and cosegregate with HSCR in at least two different pedigrees. Since HSCR, MEN2A, and MEN2B represent defects of neural crest cell development, we hypothesize that they originate from mutations in different genes clustered in the centromeric region of 10q.

Low-copy repeats on chromosome 22q11.2 show replication timing switches, DNA flexibility peaks and stress inducible asynchrony, sharing instability features with fragile sites

The 22q11.2 region is a hotspot for chromosomal rearrangements mediated by LCR22A-D low-copy repeats. Sequence motifs and homology-driven mechanisms have been suggested to mediate rearrangements. Nevertheless, recent evidence has emphasized the role of functional properties in genome instability, suggesting that replication timing transition regions could be peculiarly prone to genetic damage. In this work, we show that an early-late replication-transition zone is localised within LCR22A, the shared proximal endpoint of the majority of deletions and duplications of 22q11.2 region. Transition zone is characterized by asynchronous replication and by a DNA flexibility peak, features which are relevant for double-strand breaks and rearrangements at fragile sites. This and other flexibility peaks, associated with less relevant replication anomalies, are present in clusters inside LCR22A, B and D. All of them are composed of modules of AT-rich sequences, DNA satellites, and a HIV-1 integration site; moreover, they have coincidental position with boundaries of duplicons inside segmental duplications and with breakpoints of recurrent translocations. Noteworthy, flexibility peaks also lay at breakpoints of translocation partner chromosomes, three of which, 1p21.2, 8q24.13 and 11q23.3, have been positioned inside known common fragile sites. In many cases peaks are associated with potential matrix attachment regions (MARs). We propose that, similarly to fragile sites, replication perturbation and flexibility peaks may mediate strand breakage and rearrangements. Consistently with this view we show that the replication timing transition zone detected inside LCR22A is susceptible to replicative stress by aphidicolin, known inducer of fragile sites. These findings emphasize the significance of mutagenic exposure for the constitutional syndrome origin.

C620R mutation of the murine ret proto-oncogene: Loss of function effect in homozygotes and possible gain of function effect in heterozygotes

Germline RET mutations are responsible for different inherited disorders: Hirschsprung disease (congenital aganglionic megacolon), caused by loss of function mutations, familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2, caused by gain of function mutations. Intriguingly, some RET mutations, including C620R, are associated with both types of diseases. To investigate the dual role of such RET mutations, a mouse model with a targeted mutation retC620R was generated. retC620R/C620R offspring die during the first postnatal day, and show kidney agenesis and intestinal aganglionosis. Decreased outgrowth of the Ret‐positive cells was observed in retC620R/C620R neuronal cell cultures, which is suggestive of an impaired migration, proliferation or survival of the Ret‐expressing cells. Electronmicroscopy revealed the absence of membrane‐bound Ret in retC620R/C620R cells as compared to ret+/+ and ret+/C620R cells. On the other hand, aged ret+/C620R mice develop precancerous lesions in the adrenal gland or in the thyroid. Our results suggest that the retC620R mutation has a loss of function effect in homozygotes and exhibits a dominant gain of function effect with low penetrance causing hyperplasia in heterozygotes.