Short-Term Striatal Gene Expression Responses to Brain-Derived Neurotrophic Factor Are Dependent on MEK and ERK Activation

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is believed to be an important regulator of striatal neuron survival, differentiation, and plasticity. Moreover, reduction of BDNF delivery to the striatum has been implicated in the pathophysiology of Huntington's disease. Nevertheless, many essential aspects of BDNF responses in striatal neurons remain to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we assessed the relative contributions of multipartite intracellular signaling pathways to the short-term induction of striatal gene expression by BDNF. To identify genes regulated by BDNF in these GABAergic cells, we first used DNA microarrays to quantify their transcriptomic responses following 3 h of BDNF exposure. The signal transduction pathways underlying gene induction were subsequently dissected using pharmacological agents and quantitative real-time PCR. Gene expression responses to BDNF were abolished by inhibitors of TrkB (K252a) and calcium (chelator BAPTA-AM and transient receptor potential cation channel [TRPC] antagonist SKF-96365). Interestingly, inhibitors of mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) and extracellular signal-regulated kinase ERK also blocked the BDNF-mediated induction of all tested BDNF-responsive genes. In contrast, inhibitors of nitric oxide synthase (NOS), phosphotidylinositol-3-kinase (PI3K), and CAMK exhibited less prevalent, gene-specific effects on BDNF-induced RNA expression. At the nuclear level, the activation of both Elk-1 and CREB showed MEK dependence. Importantly, MEK-dependent activation of transcription was shown to be required for BDNF-induced striatal neurite outgrowth, providing evidence for its contribution to striatal neuron plasticity. CONCLUSIONS: These results show that the MEK/ERK pathway is a major mediator of neuronal plasticity and other important BDNF-dependent striatal functions that are fulfilled through the positive regulation of gene expression

Transcriptional dysregulation in Huntington's disease

Huntington's disease (HD) is a progressive neurodegenerative disorder with autosomal dominant inheritance. It is caused by a singular mutation in exon 1 of the HD gene encoding an abnormal polyglutamine (polyQ) expansion in the N-terminal region of the huntingtin (htt) protein. HD is associated with severe motor, cognitive and psychiatric symptoms, and death occurs within one to two decades. Postmortem analysis of the brains of affected patients reveals proteinaceous inclusions, marked striatal atrophy, and extensive transcriptional dysregulation in the caudate, putamen and cerebral cortex. Both wild-type and mutant htt physically interact with numerous transcription factors, coactivators and repressors, implying aberrant protein-protein interactions and sequestration as a possible cause for the manifest gene expression alterations. The well-established and robust gene expression changes in the HD brain motivated our investigation of potential transcriptomic biomarkers in peripheral blood. The ultimate aim was to identify surrogate markers which can discriminate between different HD states, and thereby be valuable in evaluating the possible effects of new candidate HD therapeutics. Chapter 5.1. describes the result of a cross-sectional analysis of human HD samples using microarray and real-time PCR techniques. We identified one RNA, IER3, which showed significantly higher expression levels in HD than control and tracked with disease progression. In contrast, potential RNA biomarkers identified in a previous study were not differentially expressed between HD and controls in our sample set. Transcriptomic changes in HD can be seen as a composite representation of distinct, parallel disease mechanisms involving both cell-autonomous and trans-cellular effects. Primary striatal neuron models of HD comprised of rat ganglionic eminence cells transduced with lentiviral expression vectors delivering different lengths and dosages of htt protein have been extensively characterized in our lab (described in chapter 5.2.). Gene expression profiling of these in vitro HD models demonstrated high concordance with human HD. Disease-related effects on the transcriptome were dependent on the length of the polyQ repeat and time of mutant htt exposure. Importantly, the observed changes occurred in the absence of brain circuitry, and also in the absence of brain-derived neurotrophic factor, indicating that the transcriptomic level effects arise from direct actions of mutant htt in medium spiny neurons. The transcriptomic signatures of cultured neurons have also been employed as readout of neuroprotective effects of sirtuin 2 (SIRT2) inhibitors (see chapter 5.3.). Sirtuins comprise Class III histone deacetylases and thus have potential gene regulatory effects through transcription factor and chromatin modifications. Biological pathway analysis within the Gene Ontology framework showed that acute treatment with novel small-molecule SIRT2 inhibitors caused the downregulation of genes associated with sterol and lipid metabolism in both wild-type and polyQ htt expressing cells. In contrast, SIRT2 inhibition did not reverse polyglutamine-induced transcriptional dysregulation, suggesting that it works through an alternate mechanism. These results build on the current evidence supporting that sirtuins comprise interesting targets for anti-neurodegenerative drug development and suggest new candidate mechanisms for their therapeutic effects in neurons

Developmental expression of Synaptotagmin isoforms in single calyx of Held-generating neurons

The large glutamatergic calyx of Held synapse in the auditory brainstem has become a powerful model for studying transmitter release mechanisms, but the molecular bases of presynaptic function at this synapse are not well known. Here, we have used single-cell quantitative PCR (qPCR) to study the developmental expression of all major Synaptotagmin (Syt) isoforms in putative calyx of Held-generating neurons (globular bushy cells) of the ventral cochlear nucleus. Using electrophysiological criteria and the expression of marker genes including VGluTs (vesicular glutamate transporters), Ca2+ binding proteins, and the transcription factor Math5, we identified a subset of the recorded neurons as putative calyx of Held-generating bushy cells. At postnatal days 12-15 these neurons expressed Syt-2 and Syt-11, and also Syt-3, -4, -7 and -13 at lower levels, whereas Syt-1 and -9 were absent. Interestingly, early in development (at P3-P6), immature bushy cells expressed a larger number of Syt-isoforms, with Syt-1, Syt-5, Syt-9 and Syt-13 detected in a significantly higher percentage of neurons. Our study sheds light on the molecular properties of putative calyx of Held-generating neurons and shows the developmental regulation of the Syt-isoform expression profile in a single neuron type. (C) 2010 Elsevier Inc. All rights reserved

Diminished activity-dependent brain-derived neurotrophic factor expression underlies cortical neuron microcircuit hypoconnectivity resulting from exposure to mutant huntingtin fragments

Although previous studies of Huntington's disease (HD) have addressed many potential mechanisms of striatal neuron dysfunction and death, it is also known, based on clinical findings, that cortical function is dramatically disrupted in HD. With respect to disease etiology, however, the specific molecular and neuronal circuit bases for the cortical effects of mutant huntingtin (htt) have remained largely unknown. In the present work, we studied the relationship between the molecular effects of mutant htt fragments in cortical cells and the corresponding behavior of cortical neuron microcircuits by using a novel cellular model of HD. We observed that a transcript-selective diminution in activity-dependent brain-derived neurotrophic factor (BDNF) expression preceded the onset of a synaptic connectivity deficit in ex vivo cortical networks, which manifested as decreased spontaneous collective burst-firing behavior measured by multielectrode array substrates. Decreased BDNF expression was determined to be a significant contributor to network-level dysfunction, as shown by the ability of exogenous BDNF to ameliorate cortical microcircuit burst firing. The molecular determinants of the dysregulation of activity-dependent BDNF expression by mutant htt seem to be distinct from previously elucidated mechanisms, because they do not involve known neuron-restrictive silencer factor/RE1-silencing transcription factor-regulated promoter sequences but instead result from dysregulation of BDNF exon IV and VI transcription. These data elucidate a novel HD-related deficit in BDNF gene regulation as a plausible mechanism of cortical neuron hypoconnectivity and cortical function deficits in HD. Moreover, the novel model paradigm established here is well suited to further mechanistic and drug screening research applications. Copyright \ua9 2010 by The American Society for Pharmacology and Experimental Therapeutics

Dysregulation of gene expression in primary neuron models of Huntington's disease shows that polyglutamine-related effects on the striatal transcriptome may not be dependent on brain circuitry

Gene expression changes are a hallmark of the neuropathology of Huntington's disease (HD), but the exact molecular mechanisms of this effect remain uncertain. Here, we report that in vitro models of disease comprised of primary striatal neurons expressing N-terminal fragments of mutant huntingtin (via lentiviral gene delivery) faithfully reproduce the gene expression changes seen in human HD. Neither viral infection nor unrelated (enhanced green fluorescent protein) transgene expression had a major effect on resultant RNA profiles. Expression of a wild-type fragment of huntingtin [htt171-18Q] also caused only a small number of RNA changes. The disease-related signal in htt171-82Q versus htt171-18Q comparisons was far greater, resulting in the differential detection of 20% of all mRNA probe sets. Transcriptomic effects of mutated htt171 are time- and polyglutamine-length dependent and occur in parallel with other manifestations of polyglutamine toxicity over 4-8 weeks. Specific RNA changes in htt171-82Q-expressing striatal cells accurately recapitulated those observed in human HD caudate and included decreases in PENK (proenkephalin), RGS4 (regulator of G-protein signaling 4), dopamine D(1) receptor (DRD1), DRD2, CNR1 (cannabinoid CB(1) receptor), and DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32; also known as PPP1R1B) mRNAs. HD-related transcriptomic changes were also observed in primary neurons expressing a longer fragment of mutant huntingtin (htt853-82Q). The gene expression changes observed in cultured striatal neurons are not secondary to abnormalities of neuronal firing or glutamatergic, dopaminergic, or brain-derived neurotrophic factor signaling, thereby demonstrating that HD-induced dysregulation of the striatal transcriptome might be attributed to intrinsic effects of mutant huntingtin

SIRT2 inhibition achieves neuroprotection by decreasing sterol biosynthesis

Huntington’s disease (HD), an incurable neurodegenerative disorder, has a complex pathogenesis including protein aggregation and the dysregulation of neuronal transcription and metabolism. Here, we demonstrate that inhibition of sirtuin 2 (SIRT2) achieves neuroprotection in cellular and invertebrate models of HD. Genetic or pharmacologic inhibition of SIRT2 in a striatal neuron model of HD resulted in gene expression changes including significant down-regulation of RNAs responsible for sterol biosynthesis. Whereas mutant huntingtin fragments increased sterols in neuronal cells, SIRT2 inhibition reduced sterol levels via decreased nuclear trafficking of SREBP-2. Importantly, manipulation of sterol biosynthesis at the transcriptional level mimicked SIRT2 inhibition, demonstrating that the metabolic effects of SIRT2 inhibition are sufficient to diminish mutant huntingtin toxicity. These data identify SIRT2 inhibition as a promising avenue for HD therapy and elucidate a unique mechanism of SIRT2-inhibitor-mediated neuroprotection. Furthermore, the ascertainment of SIRT2’s role in regulating cellular metabolism demonstrates a central function shared with other sirtuin proteins

SIRT2 inhibition achieves neuroprotection by decreasing sterol biosynthesis.

Huntington's disease (HD), an incurable neurodegenerative disorder, has a complex pathogenesis including protein aggregation and the dysregulation of neuronal transcription and metabolism. Here, we demonstrate that inhibition of sirtuin 2 (SIRT2) achieves neuroprotection in cellular and invertebrate models of HD. Genetic or pharmacologic inhibition of SIRT2 in a striatal neuron model of HD resulted in gene expression changes including significant down-regulation of RNAs responsible for sterol biosynthesis. Whereas mutant huntingtin fragments increased sterols in neuronal cells, SIRT2 inhibition reduced sterol levels via decreased nuclear trafficking of SREBP-2. Importantly, manipulation of sterol biosynthesis at the transcriptional level mimicked SIRT2 inhibition, demonstrating that the metabolic effects of SIRT2 inhibition are sufficient to diminish mutant huntingtin toxicity. These data identify SIRT2 inhibition as a promising avenue for HD therapy and elucidate a unique mechanism of SIRT2-inhibitor-mediated neuroprotection. Furthermore, the ascertainment of SIRT2's role in regulating cellular metabolism demonstrates a central function shared with other sirtuin proteins

SIRT2- and NRF2-Targeting Thiazole-Containing Compound with Therapeutic Activity in Huntington's Disease Models.

There are currently no disease-modifying therapies for the neurodegenerative disorder Huntington's disease (HD). This study identified novel thiazole-containing inhibitors of the deacetylase sirtuin-2 (SIRT2) with neuroprotective activity in ex vivo brain slice and Drosophila models of HD. A systems biology approach revealed an additional SIRT2-independent property of the lead-compound, MIND4, as an inducer of cytoprotective NRF2 (nuclear factor-erythroid 2 p45-derived factor 2) activity. Structure-activity relationship studies further identified a potent NRF2 activator (MIND4-17) lacking SIRT2 inhibitory activity. MIND compounds induced NRF2 activation responses in neuronal and non-neuronal cells and reduced production of reactive oxygen species and nitrogen intermediates. These drug-like thiazole-containing compounds represent an exciting opportunity for development of multi-targeted agents with potentially synergistic therapeutic benefits in HD and related disorders