Luis Tecedor

Structure and activity of putative intronic miRNA promoters

MicroRNAs (miRNAs) are RNA sequences of approximately 22 nucleotides that mediate post-transcriptional regulation of specific mRNAs. miRNA sequences are dispersed throughout the genome and are classified as intergenic (between genes) or intronic (embedded into a gene). Intergenic miRNAs are expressed by their own promoter, and until recently, it was supposed that intronic miRNAs are transcribed from their host gene. Here, we performed a genomic analysis of currently known intronic miRNA regions and observed that approximately 35% of intronic miRNAs have upstream regulatory elements consistent with promoter function. Among all intronic miRNAs, 30% have associated Pol II regulatory elements, including transcription start sites, CpG islands, expression sequence tags, and conserved transcription factor binding sites, while 5% contain RNA Pol III regulatory elements (A/B box sequences). We cloned intronic regions encompassing miRNAs and their upstream Pol II (miR-107, miR-126, miR-208b, miR-548f-2, miR-569, and miR-590) or Pol III (miR-566 and miR-128-2) sequences into a promoterless plasmid, and confirmed that miRNA expression occurs independent of host gene transcription. For miR-128-2, a miRNA overexpressed in acute lymphoblastic leukemia, ChIP analysis suggests dual regulation by both intronic (Pol III) and host gene (Pol II) promoters. These data support complex regulation of intronic miRNA expression, and have relevance to disregulation in disease settings.

Clarifying lysosomal storage diseases

Lysosomal storage diseases (LSDs) are a class of metabolic disorders caused by mutations in proteins critical for lysosomal function. Such proteins include lysosomal enzymes, lysosomal integral membrane proteins, and proteins involved in the post-translational modification and trafficking of lysosomal proteins. There are many recognized forms of LSDs and, although individually rare, their combined prevalence is estimated to be 1 in 8000 births. Over two-thirds of LSDs involve central nervous system (CNS) dysfunction (progressive cognitive and motor decline) and these symptoms are often the most debilitating. Although the genetic basis for these disorders is clear and the biochemistry of the proteins well understood, the cellular mechanisms by which deficiencies in these proteins disrupt neuronal viability remain ambiguous. In this review, we provide an overview of the widespread cellular perturbations occurring in LSDs, how they might be linked and interventions that may specifically or globally correct those defects.

Reinstating Aberrant mTORC1 Activity in Huntington’s Disease Mice Improves Disease Phenotypes

Huntingtons disease (HD) is caused by a polyglutamine tract expansion in huntingtin (HTT). Despite HTTs ubiquitous expression, there is early and robust vulnerability in striatum, the cause of which is poorly understood. Here, we provide evidence that impaired striatal mTORC1 activity underlies varied metabolic and degenerative phenotypes in HD brain and show that introducing the constitutively active form of the mTORC1 regulator, Rheb, into HD mouse brain, alleviates mitochondrial dysfunction, aberrant cholesterol homeostasis, striatal atrophy, impaired dopamine signaling, and increases autophagy. We also find that the expression of Rhes, a striatum-enriched mTOR activator, is reduced in HD patient and mouse brain and that exogenous addition of Rhes alleviates motor deficits and improves brain pathology in HD mice. Our combined work indicates that impaired Rhes/mTORC1 activity in HD brain may underlie the notable striatal susceptibility and thus presents a promising therapeutic target for HD therapy.

Dicer is required for proliferation, viability, migration and differentiation in corticoneurogenesis.

In mice, microRNAs (miRNAs) are required for embryonic viability, and previous reports implicate miRNA participation in brain cortical neurogenesis. Here, we provide a more comprehensive analysis of miRNA involvement in cortical brain development. To accomplish this we used mice in which Dicer, the RNase III enzyme necessary for canonical miRNA biogenesis, is depleted from Nestin-expressing progenitors and progeny cells. We systematically assessed how Dicer depletion impacts proliferation, cell death, migration and differentiation in the developing brain. Using markers for proliferation and in vivo labeling with thymidine analogs, we found reduced numbers of proliferating cells, and altered cell cycle kinetics from embryonic day 15.5 (E15.5). Progenitor cells were distributed aberrantly throughout the cortex rather than restricted to the ventricular and subventricular zones. Activated Caspase3 was elevated, reflecting increased cortical cell death as early as E15.5. Cajal-Retzius-positive cells were more numerous at E15.5 and were dysmorphic relative to control cortices. Consistent with this, Reelin levels were enhanced. Doublecortin and Rnd2 were also increased and showed altered distribution, supporting a strong regulatory role for miRNAs in both early and late neuronal migration. In addition, GFAP staining at E15.5 was more intense and disorganized throughout the cortex with Dicer depletion. These results significantly extend earlier works, and emphasize the impact of miRNAs on neural progenitor cell proliferation, apoptosis, migration, and differentiation in the developing mammalian brain.

A Knock-In Reporter Model of Batten Disease

Juvenile neuronal ceroid lipofuscinosis is a severe inherited neurodegenerative disease resulting from mutations in CLN3 (ceroid-lipofuscinosis, neuronal 3, juvenile). CLN3 function, and where and when it is expressed during development, is not known. In this study, we generated a knock-in reporter mouse to elucidate CLN3 expression during embryogenesis and after birth and to correlate expression and behavior in a CLN3-deficient mouse. In embryonic brain, expression appeared in the cortical plate. In postnatal brain, expression was prominent in the cortex, subiculum, parasubiculum, granule neurons of the dentate gyrus, and some brainstem nuclei. In adult brain, reporter gene expression waned in most areas but remained in vascular endothelia and the dentate gyrus. Mice homozygous for Cln3 deletion showed two hallmark pathological features of the neuronal ceroid lipofuscinosises: autofluorescent inclusions and lysosomal enzyme elevation. Moreover, CLN3-deficient reporter mice displayed progressive neurological deficits, including impaired motor function, decreased overall activity, acquisition of resting tremors, and increased susceptibility to pentilentetrazole-induced seizures. Notably, seizure induction in heterozygous mice was accompanied by enhanced reporter expression. This model provides us with the unique ability to correlate expression with pathology and behavior, thus facilitating the elucidation of CLN3 function and the pathogenesis of Batten disease.

AAV gene transfer delays disease onset in a TPP1-deficient canine model of the late infantile form of Batten disease

AAV-mediated gene transfer to ependymal cells in a dog model of Batten disease provides sustained enzyme replacement and delays disease onset. Gene therapy for Batten disease Repeated enzyme replacement therapy via infusion of the enzyme into blood vessels is an established treatment modality for many lysosomal storage diseases but not for those with significant brain involvement. In new work, Katz and colleagues test gene delivery of the missing enzyme to cells that line the fluid-filled spaces of the brain and measure secretion of the enzyme into the cerebrospinal fluid. Using a dog model of a lysosomal storage disease, they show that this strategy delayed disease onset, extended life span, and protected dogs from early cognitive decline, suggesting that this approach could improve the lives of children suffering from the same or similar diseases. The most common form of the childhood neurodegenerative disease late infantile neuronal ceroid lipofuscinosis (also called Batten disease) is caused by deficiency of the soluble lysosomal enzyme tripeptidyl peptidase 1 (TPP1) resulting from mutations in the TPP1 gene. We tested whether TPP1 gene transfer to the ependyma, the epithelial lining of the brain ventricular system, in TPP1-deficient dogs would be therapeutically beneficial. A one-time administration of recombinant adeno-associated virus (rAAV) expressing canine TPP1 (rAAV.caTPP1) resulted in high expression of TPP1 predominantly in ependymal cells and secretion of the enzyme into the cerebrospinal fluid leading to clinical benefit. Diseased dogs treated with rAAV.caTPP1 showed delays in onset of clinical signs and disease progression, protection from cognitive decline, and extension of life span. By immunostaining and enzyme assay, recombinant protein was evident throughout the brain and spinal cord, with correction of the neuropathology characteristic of the disease. This study in a naturally occurring canine model of TPP1 deficiency highlights the utility of AAV transduction of ventricular lining cells to accomplish stable secretion of recombinant protein for broad distribution in the central nervous system and therapeutic benefit.

CLN3 Loss Disturbs Membrane Microdomain Properties and Protein Transport in Brain Endothelial Cells

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal childhood-onset neurodegenerative disorder caused by mutations in ceroid lipofuscinosis neuronal-3 (CLN3), a hydrophobic transmembrane protein of unresolved function. Previous studies indicate blood–brain barrier (BBB) defects in JNCL, and our earlier report showed prominent Cln3 expression in mouse brain endothelium. Here we find that CLN3 is necessary for normal trafficking of the microdomain-associated proteins caveolin-1, syntaxin-6, and multidrug resistance protein 1 (MDR1) in brain endothelial cells. Correspondingly, CLN3-null cells have reduced caveolae, and impaired caveolae- and MDR1-related functions including endocytosis, drug efflux, and cell volume regulation. We also detected an abnormal blood–brain barrier response to osmotic stress in vivo. Evaluation of the plasma membrane with fluorescent sphingolipid probes suggests microdomain destabilization and enhanced fluidity in CLN3-null cells. In further work we found that application of the glycosphingolipid lactosylceramide to CLN3-deficient cells rescues protein transport and caveolar endocytosis. Last, we show that CLN3 localizes to the trans-Golgi network (TGN) and partitions with buoyant microdomain fractions. We propose that CLN3 facilitates TGN-to-plasma membrane transport of microdomain-associated proteins. Insult to this pathway may underlie BBB dysfunction and contribute to JNCL pathogenesis.

A knock-in reporter mouse model for Batten disease reveals predominant expression of Cln3 in visual, limbic and subcortical motor structures.

Juvenile neuronal ceroid lipofuscinosis (JNCL) or Batten disease is an autosomal recessive neurodegenerative disorder of children caused by mutation in CLN3. JNCL is characterized by progressive visual impairment, cognitive and motor deficits, seizures and premature death. Information about the localization of CLN3 expressing neurons in the nervous system is limited, especially during development. The present study has systematically mapped the spatial and temporal localization of CLN3 reporter neurons in the entire nervous system including retina, using a knock-in reporter mouse model. CLN3 reporter is expressed predominantly in post-migratory neurons in visual and limbic cortices, anterior and intralaminar thalamic nuclei, amygdala, cerebellum, red nucleus, reticular formation, vestibular nuclei and retina. CLN3 reporter in the nervous system is mainly expressed during the first postnatal month except in the dentate gyrus, parasolitary nucleus and retina, where it is still strongly expressed in adulthood. The predominant distribution of CLN3 reporter neurons in visual, limbic and subcortical motor structures correlates well with the clinical symptoms of JNCL. These findings have also revealed potential target brain regions and time periods for future investigations of the disease mechanisms and therapeutic intervention.

CLN3 deficient cells display defects in the ARF1-Cdc42 pathway and actin-dependent events.

Juvenile Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL) is a devastating neurodegenerative disease caused by mutations in CLN3, a protein of undefined function. Cell lines derived from patients or mice with CLN3 deficiency have impairments in actin-regulated processes such as endocytosis, autophagy, vesicular trafficking, and cell migration. Here we demonstrate the small GTPase Cdc42 is misregulated in the absence of CLN3, and thus may be a common link to multiple cellular defects. We discover that active Cdc42 (Cdc42-GTP) is elevated in endothelial cells from CLN3 deficient mouse brain, and correlates with enhanced PAK-1 phosphorylation, LIMK membrane recruitment, and altered actin-driven events. We also demonstrate dramatically reduced plasma membrane recruitment of the Cdc42 GTPase activating protein, ARHGAP21. In line with this, GTP-loaded ARF1, an effector of ARHGAP21 recruitment, is depressed. Together these data implicate misregulated ARF1-Cdc42 signaling as a central defect in JNCL cells, which in-turn impairs various cell functions. Furthermore our findings support concerted action of ARF1, ARHGAP21, and Cdc42 to regulate fluid phase endocytosis in mammalian cells. The ARF1-Cdc42 pathway presents a promising new avenue for JNCL therapeutic development.

Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo

The neuronal ceroid lipofuscinoses are a class of inherited neurodegenerative diseases characterized by the accumulation of autofluorescent storage material. The most common neuronal ceroid lipofuscinosis has juvenile onset with rapid onset blindness and progressive degeneration of cognitive processes. The juvenile form is caused by mutations in the CLN3 gene, which encodes the protein CLN3. While mouse models of Cln3 deficiency show mild disease phenotypes, it is apparent from patient tissue- and cell-based studies that its loss impacts many cellular processes. Using Cln3 deficient mice, we previously described defects in mouse brain endothelial cells and blood-brain barrier (BBB) permeability. Here we expand on this to other components of the BBB and show that Cln3 deficient mice have increased astrocyte endfeet area. Interestingly, this phenotype is corrected by treatment with a commonly used GAP junction inhibitor, carbenoxolone (CBX). In addition to its action on GAP junctions, CBX has also been proposed to alter lipid microdomains. In this work, we show that CBX modifies lipid microdomains and corrects membrane fluidity alterations in Cln3 deficient endothelial cells, which in turn improves defects in endocytosis, caveolin-1 distribution at the plasma membrane, and Cdc42 activity. In further work using the NIH Library of Integrated Network-based Cellular Signatures (LINCS), we discovered other small molecules whose impact was similar to CBX in that they improved Cln3-deficient cell phenotypes. Moreover, Cln3 deficient mice treated orally with CBX exhibited recovery of impaired BBB responses and reduced auto-fluorescence. CBX and the compounds identified by LINCS, many of which have been used in humans or approved for other indications, may find therapeutic benefit in children suffering from CLN3 deficiency through mechanisms independent of their original intended use.