Colleen M. Dewey

Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes

TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)n is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)nTA(TG)m, is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.

TDP-43 is a developmentally-regulated protein essential for early embryonic development

TDP-43 is a DNA/RNA-binding protein implicated in multiple steps of transcriptional and post-transcriptional regulation of gene expression. Alteration of this multifunctional protein is associated with a number of neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin positive inclusions. Whereas a pathological link to neurodegenerative disorders has been established, the cellular and physiological functions of TDP-43 remain unknown. In this study, we show that TDP-43 is a nuclear protein with persistent high-level expression during embryonic development and with progressively decreased protein levels during postnatal development. In mice where the TDP-43 gene (Tardbp) was disrupted using a gene trap that carries a β-galactosidase marker gene, heterozygous (Tardbp+/−) mice are fertile and healthy, but intercrosses of Tardbp+/− mice yielded no viable homozygotic null (Tardbp−/−) mice. Indeed, Tardbp−/− embryos die between 3.5 and 8.5 days of development. Tardbp−/− blastocysts grown in cell culture display abnormal expansion of their inner cell mass. The pattern of β-galactosidase staining at E9.5 Tardbp+/− embryos is predominantly restricted to the neuroepithelium and remains prominent in neural progenitors at E10.5–12.5. TDP-43 is detected in spinal cord progenitors and in differentiated motor neurons as well as in the dorsal root ganglia at E12.5. β-Galactosidase staining of tissues from adult Tardbp+/− mice shows widespread expression of TDP-43, including prominent levels in various regions of the central nervous system afflicted in neurodegenerative disorders. These results indicate that TDP-43 is developmentally regulated and indispensible for early embryonic development.

Cocaine Regulates MEF2 to Control Synaptic and Behavioral Plasticity

Repeated exposure to cocaine causes sensitized behavioral responses and increased dendritic spines on medium spiny neurons of the nucleus accumbens (NAc). We find that cocaine regulates myocyte enhancer factor 2 (MEF2) transcription factors to control these two processes in vivo. Cocaine suppresses striatal MEF2 activity in part through a mechanism involving cAMP, the regulator of calmodulin signaling (RCS), and calcineurin. We show that reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, we find that increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, our findings implicate MEF2 as a key regulator of structural synapse plasticity and sensitized responses to cocaine and suggest that reducing MEF2 activity (and increasing spine density) in NAc may be a compensatory mechanism to limit long-lasting maladaptive behavioral responses to cocaine.

Subunit-Specific Regulation of NMDA Receptor Endocytosis

At excitatory synapses, both NMDA and AMPA receptors are localized to the postsynaptic density (PSD). However, unlike AMPA receptors, synaptic NMDA receptors are stable components of the PSD. Even so, surface-expressed NMDA receptors undergo endocytosis, which is more robust early in development and declines during synaptic development. We investigated the subunit-specific contributions to NMDA receptor endocytosis, specifically defining the endocytic motifs and endocytic pathways preferred by the NR2A and NR2B subunits. We find that NR2A and NR2B have distinct endocytic motifs encoded in their distal C termini and that these interact with clathrin adaptor complexes with differing affinities. We also find that NR2A and NR2B sort into different intracellular pathways after endocytosis, with NR2B preferentially trafficking through recycling endosomes. In mature cultures, we find that NR2B undergoes more robust endocytosis than NR2A, consistent with previous studies showing that NR2A is more highly expressed at stable synaptic sites. Our findings demonstrate fundamental differences between NR2A and NR2B that help clarify developmental changes in NMDA receptor trafficking and surface expression.

TDP-43 is directed to stress granules by sorbitol, a novel physiological osmotic and oxidative stressor

ABSTRACT TDP-43, or TAR DNA-binding protein 43, is a pathological marker of a spectrum of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. TDP-43 is an RNA/DNA-binding protein implicated in transcriptional and posttranscriptional regulation. Recent work also suggests that TDP-43 associates with cytoplasmic stress granules, which are transient structures that form in response to stress. In this study, we establish sorbitol as a novel physiological stressor that directs TDP-43 to stress granules in Hek293T cells and primary cultured glia. We quantify the association of TDP-43 with stress granules over time and show that stress granule association and size are dependent on the glycine-rich region of TDP-43, which harbors the majority of pathogenic mutations. Moreover, we establish that cells harboring wild-type and mutant TDP-43 have distinct stress responses: mutant TDP-43 forms significantly larger stress granules, and is incorporated into stress granules earlier, than wild-type TDP-43; in striking contrast, wild-type TDP-43 forms more stress granules over time, but the granule size remains relatively unchanged. We propose that mutant TDP-43 alters stress granule dynamics, which may contribute to the progression of TDP-43 proteinopathies.

Loss of Predominant Shank3 Isoforms Results in Hippocampus-Dependent Impairments in Behavior and Synaptic Transmission

The Shank3 gene encodes a scaffolding protein that anchors multiple elements of the postsynaptic density at the synapse. Previous attempts to delete the Shank3 gene have not resulted in a complete loss of the predominant naturally occurring Shank3 isoforms. We have now characterized a homozygous Shank3 mutation in mice that deletes exon 21, including the Homer binding domain. In the homozygous state, deletion of exon 21 results in loss of the major naturally occurring Shank3 protein bands detected by C-terminal and N-terminal antibodies, allowing us to more definitively examine the role of Shank3 in synaptic function and behavior. This loss of Shank3 leads to an increased localization of mGluR5 to both synaptosome and postsynaptic density-enriched fractions in the hippocampus. These mice exhibit a decrease in NMDA/AMPA excitatory postsynaptic current ratio in area CA1 of the hippocampus, reduced long-term potentiation in area CA1, and deficits in hippocampus-dependent spatial learning and memory. In addition, these mice also exhibit motor-coordination deficits, hypersensitivity to heat, novelty avoidance, altered locomotor response to novelty, and minimal social abnormalities. These data suggest that Shank3 isoforms are required for normal synaptic transmission/plasticity in the hippocampus, as well as hippocampus-dependent spatial learning and memory.

Suberoylanilide Hydroxamic Acid (Vorinostat) Up-regulates Progranulin Transcription RATIONAL THERAPEUTIC APPROACH TO FRONTOTEMPORAL DEMENTIA

Progranulin (GRN) haploinsufficiency is a frequent cause of familial frontotemporal dementia, a currently untreatable progressive neurodegenerative disease. By chemical library screening, we identified suberoylanilide hydroxamic acid (SAHA), a Food and Drug Administration-approved histone deacetylase inhibitor, as an enhancer of GRN expression. SAHA dose-dependently increased GRN mRNA and protein levels in cultured cells and restored near-normal GRN expression in haploinsufficient cells from human subjects. Although elevation of secreted progranulin levels through a post-transcriptional mechanism has recently been reported, this is, to the best of our knowledge, the first report of a small molecule enhancer of progranulin transcription. SAHA has demonstrated therapeutic potential in other neurodegenerative diseases and thus holds promise as a first generation drug for the prevention and treatment of frontotemporal dementia.

Expression, localization, and biochemical characterization of nicotinamide mononucleotide adenylyltransferase 2.

Nicotinamide mononucleotide (NMN) adenylyltransferase 2 (Nmnat2) catalyzes the synthesis of NAD from NMN and ATP. The Nmnat2 transcript is expressed predominately in the brain; we report here that Nmnat2 is a low abundance protein expressed in neurons. Previous studies indicate that Nmnat2 localizes to Golgi. As Nmnat2 is not predicted to contain a signal sequence, lipid-binding domain, or transmembrane domain, we investigated the nature of this interaction. These experiments reveal that Nmnat2 is palmitoylated in vitro, and this modification is required for membrane association. Surprisingly, exogenous Nmnat2 is toxic to neurons, indicating that protein levels must be tightly regulated. To analyze Nmnat2 localization in neurons (previous experiments relied on exogenous expression in HeLa cells), mouse brains were fractionated, showing that Nmnat2 is enriched in numerous membrane compartments including synaptic terminals. In HeLa cells, in addition to Golgi, Nmnat2 localizes to Rab7-containing late endosomes. These studies show that Nmnat2 is a neuronal protein peripherally attached to membranes via palmitoylation and suggest that Nmnat2 is transported to synaptic terminals via an endosomal pathway.

γ-Secretase-regulated Proteolysis of the Notch Receptor by Mitochondrial Intermediate Peptidase

Notch is a transmembrane receptor that controls a diverse array of cellular processes including cell proliferation, differentiation, survival, and migration. The cellular outcome of Notch signaling is dependent on extracellular and intracellular signals, but the complexities of its regulation are not well understood. Canonical Notch signaling involves ligand association that triggers sequential and regulated proteolysis of Notch at several sites. Ligand-dependent proteolysis at the S2 site removes the bulk of the extracellular domain of Notch. Subsequent γ-secretase-mediated intramembrane proteolysis of the remaining membrane-tethered Notch fragment at the S3 site produces a nuclear-destined Notch intracellular domain (NICD). Here we show that following γ-secretase cleavage, Notch is proteolyzed at a novel S5 site. We have identified this S5 site to be eight amino acids downstream of the S3 site. Biochemical fractionation and purification resulted in the identification of the S5 site protease as the mitochondrial intermediate peptidase (MIPEP). Expression of the MIPEP-cleaved NICD (ΔNICD) results in a decrease in cell viability and mitochondria membrane potential. The sequential and regulated proteolysis by γ-secretase and MIPEP suggests a new means by which Notch function can be modulated.