Gloria Cantero

U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’s disease

Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer’s disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.

Cysteine String Protein- Prevents Activity-Dependent Degeneration in GABAergic Synapses

The continuous release of neurotransmitter could be seen to place a persistent burden on presynaptic proteins, one that could compromise nerve terminal function. This supposition and the molecular mechanisms that might protect highly active synapses merit investigation. In hippocampal cultures from knock-out mice lacking the presynaptic cochaperone cysteine string protein-α (CSP-α), we observe progressive degeneration of highly active synaptotagmin 2 (Syt2)-expressing GABAergic synapses, but surprisingly not of glutamatergic terminals. In CSP-α knock-out mice, synaptic degeneration of basket cell terminals occurs in vivo in the presence of normal glutamatergic synapses onto dentate gyrus granule cells. Consistent with this, in hippocampal cultures from these mice, the frequency of miniature IPSCs, caused by spontaneous GABA release, progressively declines, whereas the frequency of miniature excitatory AMPA receptor-mediated currents (mEPSCs), caused by spontaneous release of glutamate, is normal. However, the mEPSC amplitude progressively decreases. Remarkably, long-term block of glutamatergic transmission in cultures lacking CSP-α substantially rescues Syt2-expressing GABAergic synapses from neurodegeneration. These findings demonstrate that elevated neural activity increases synapse vulnerability and that CSP-α is essential to maintain presynaptic function under a physiologically high-activity regimen.

Minimal ureagenesis is necessary for survival in the murine model of hyperargininemia treated by AAV-based gene therapy

Hyperammonemia is less severe in arginase 1 deficiency compared with other urea cycle defects. Affected patients manifest hyperargininemia and infrequent episodes of hyperammonemia. Patients typically suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation, seizures and intellectual disability; death is less common than with other urea cycle disorders. In a mouse model of arginase I deficiency, the onset of symptoms begins with weight loss and gait instability, which progresses toward development of tail tremor with seizure-like activity; death typically occurs at about 2 weeks of life. Adeno-associated viral vector gene replacement strategies result in long-term survival of mice with this disorder. With neonatal administration of vector, the viral copy number in the liver greatly declines with hepatocyte proliferation in the first 5 weeks of life. Although the animals do survive, it is not known from a functional standpoint how well the urea cycle is functioning in the adult animals that receive adeno-associated virus. In these studies, we administered [1-13C] acetate to both littermate controls and adeno-associated virus-treated arginase 1 knockout animals and examined flux through the urea cycle. Circulating ammonia levels were mildly elevated in treated animals. Arginine and glutamine also had perturbations. Assessment 30 min after acetate administration demonstrated that ureagenesis was present in the treated knockout liver at levels as low at 3.3% of control animals. These studies demonstrate that only minimal levels of hepatic arginase activity are necessary for survival and ureagenesis in arginase-deficient mice and that this level of activity results in control of circulating ammonia. These results may have implications for potential therapy in humans with arginase deficiency.

Rescue of the Functional Alterations of Motor Cortical Circuits in Arginase Deficiency by Neonatal Gene Therapy

Arginase 1 deficiency is a urea cycle disorder associated with hyperargininemia, spastic diplegia, loss of ambulation, intellectual disability, and seizures. To gain insight on how loss of arginase expression affects the excitability and synaptic connectivity of the cortical neurons in the developing brain, we used anatomical, ultrastructural, and electrophysiological techniques to determine how single-copy and double-copy arginase deletion affects cortical circuits in mice. We find that the loss of arginase 1 expression results in decreased dendritic complexity, decreased excitatory and inhibitory synapse numbers, decreased intrinsic excitability, and altered synaptic transmission in layer 5 motor cortical neurons. Hepatic arginase 1 gene therapy using adeno-associated virus rescued nearly all these abnormalities when administered to neonatal homozygous knock-out animals. Therefore, gene therapeutic strategies can reverse physiological and anatomical markers of arginase 1 deficiency and therefore may be of therapeutic benefit for the neurological disabilities in this syndrome. SIGNIFICANCE STATEMENT These studies are one of the few investigations to try to understand the underlying neurological dysfunction that occurs in urea cycle disorders and the only to examine arginase deficiency. We have demonstrated by multiple modalities that, in murine layer 5 cortical neurons, a gradation of abnormalities exists based on the functional copy number of arginase: intrinsic excitability is altered, there is decreased density in asymmetrical and perisomatic synapses, and analysis of the dendritic complexity is lowest in the homozygous knock-out. With neonatal administration of adeno-associated virus expressing arginase, there is near-total recovery of the abnormalities in neurons and cortical circuits, supporting the concept that neonatal gene therapy may prevent the functional abnormalities that occur in arginase deficiency.

Development of operational immunologic tolerance with neonatal gene transfer in nonhuman primates: preliminary studies

Achieving persistent expression is a prerequisite for effective genetic therapies for inherited disorders. These proof-of-concept studies focused on adeno-associated virus (AAV) administration to newborn monkeys. Serotype rh10 AAV expressing ovalbumin and green fluorescent protein (GFP) was administered intravenously at birth and compared with vehicle controls. At 4 months postnatal age, a second injection was administered intramuscularly, followed by vaccination at 1 year of age with ovalbumin and GFP. Ovalbumin was highest 2 weeks post administration in the treated monkey, which declined but remained detectable thereafter; controls demonstrated no expression. Long-term AAV genome copies were present in myocytes. At 4 weeks, neutralizing antibodies to rh10 were present in the experimental animal only. With AAV9 administration at 4 months, controls showed transient ovalbumin expression that disappeared with the development of strong anti-ovalbumin and anti-GFP antibodies. In contrast, increased and maintained ovalbumin expression was noted in the monkey administered AAV at birth, without antibody development. After vaccination, the experimental monkey maintained levels of ovalbumin without antibodies, whereas controls demonstrated high levels of antibodies. These preliminary studies suggest that newborn AAV administration expressing secreted and intracellular xenogenic proteins may result in persistent expression in muscle, and subsequent vector administration can result in augmented expression without humoral immune responses.

The high rate of endoreduplication in the repair deficient CHO mutant EM9 parallels a reduced level of methylated deoxycytidine in DNA

It has been recently proposed that hypomethylation of DNA induced by 5-azacytidine (5-azaC) leads to reduced chromatid decatenation that ends up in endoreduplication, most likely due to a failure in topo II function [S. Mateos, I. Domínguez, N. Pastor, G. Cantero, F. Cortés, The DNA demethylating 5-azaC induces endoreduplication in cultured Chinese hamster cells, Mutat. Res. 578 (2005) 33-42]. The Chinese hamster mutant cell line EM9 has a high spontaneous frequency of endoreduplication as compared to its parental line AA8. In order to see if this is related to the degree of DNA methylation, we have investigated the basal levels of both endpoints in AA8 and EM9, as well as the effect of extensive 5-azaC-induced demethylation on the production of endoreduplication. Based on the correlation between the levels of DNA methylation and indices of endoreduplication we propose that genomic DNA hypomethylation in EM9 cell line is probably an important factor that bears significance in relation to the high basal level of endoreduplication observed in this cell line.

45. Rescue of the Functional Alterations of Motor Cortical Circuits in Arginase 1 Deficiency with AAV-Based Gene Therapy

The urea cycle is the main mechanism for terrestrial mammals to detoxify excess nitrogen. Disorders of the proximal urea cycle characteristically have periodic episodes of hyperammonemia leading to often severe and permanent neurological deterioration & disability. Ammonia has been implicated by compromising potassium buffering of astrocytic membranes and causing clinical neurological abnormalities by impairing cortical inhibition. Complete arginase 1 (Arg1) deficiency, a distal urea cycle disorder, is the least severe of these abnormalities, demonstrating neurological impairment including spasticity, loss of ambulation and seizures; while characterized by the presence of hyperargininemia, hyperammonemia is not a frequent clinical finding. While mortality is unfortunately common due to acute episodes of hyperammonemia in proximal urea cycle disorders, patients with hyperargininemia often are long-lived, however, suffering from progressive intellectual disability and spastic diplegia, and the mechanisms underlying the neurological dysfunction are not understood. To gain better insight on how the loss of arginase expression causes dysfunction in the developing brain, and if gene therapy could prevent these abnormalities, we studied how the excitability and functional and anatomical connectivity of motor cortical neurons are altered in the disorder using the murine knockout model. In addition, we examined if AAV expressing Arg1, administered IV on postnatal day 2, could rescue these findings. Results: Single- and double-copy loss of Arg1 caused dose-dependent decreases in intrinsic excitability, dendritic arborization complexity, and synapses in motor cortex layer V neurons. These findings show that 1) the intrinsic excitability of neurons of homozygous Arg1 knockout mice is abnormal and that, unexpectedly, heterozygous neurons (single copy loss) exhibit an intermediate phenotype compared to wild type and homozygous knockouts (double copy loss) (Fig. AFig. A); corresponding loss of Arg1 decreased the frequency of miniature excitatory postsynaptic currents and the amplitude of miniature inhibitory postsynaptic currents; 2) neuronal branching and spine phenotypes differ between genotypes with, unexpectedly, an intermediate phenotype for heterozygotes (Fig. BFig. B); and 3) with electron microscopic analysis and comparison of layer V synapses from arginase 1 knockout, heterozygous, and wild type mice, there is a very low density of excitatory (i.e. asymmetrical) synapses (Fig. CFig. C) in the knockout and decreased number of inhibitory (perisomatic) synapses (i.e. symmetrical) on somata of pyramidal cells, both dramatic findings. Finally, changes in synaptic morphology and abnormal ultrastructural features were found in knockout mice, also suggesting neuronal degeneration and inflammation. Neonatal intravenous administration on the second postnatal day with AAV expressing arginase 1 by a hepatocyte-specific promoter led to a near-resolution of these abnormalities when administered to homozygous Arg1 knockout animals. Summary: Our studies suggest that arginase 1 deficiency leads to severe and specific changes to intrinsic excitability and synaptic connectivity of motor cortical circuits. Importantly, we find that neonatal AAV-based Arg1 gene expression is effective in reversing both the physiological and anatomical hallmarks of the disorder.View Large Image | Download PowerPoint Slide