Luisa Quinti

Optical Visualization of Cathepsin K Activity in Atherosclerosis With a Novel, Protease-Activatable Fluorescence Sensor

Background— Cathepsin K (CatK), a potent elastinolytic and collagenolytic cysteine protease, likely participates in the evolution and destabilization of atherosclerotic plaques. To assess better the biology of CatK activity in vivo, we developed a novel near-infrared fluorescence (NIRF) probe for imaging of CatK and evaluated it in mouse and human atherosclerosis. Methods and Results— The NIRF imaging agent consists of the CatK peptide substrate GHPGGPQGKC-NH2 linked to an activatable fluorogenic polymer. In vitro, CatK produced a 2- to 14-fold activation of the agent over other cysteine and matrix metalloproteinases (P<0.0001), as well as a >8-fold activation over a control imaging agent (P<0.001). Optical imaging of atheroma revealed >100% NIRF signal increases in apolipoprotein E−/− mice in vivo (n=13; P<0.05, CatK imaging agent versus control agent) and in human carotid endarterectomy specimens ex vivo (n=14; P<0.05). Fluorescence microscopy of plaque sections demonstrated that enzymatically active CatK (positive NIRF signal) localized primarily in the vicinity of CatK-positive macrophages. Augmented NIRF signal (reflecting CatK activity) colocalized with disrupted elastin fibers within the media underlying plaques. Conclusions— Use of this novel protease-activatable NIRF agent for optical imaging in vivo demonstrated preferential localization of enzymatically active CatK to macrophages, consistent with their known greater elastinolytic capabilities compared with smooth muscle cells. Augmented CatK proteolysis in atheromata further links CatK to vascular remodeling and plaque vulnerability.

The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS

Sirtuin 2 (SIRT2) is one of seven known mammalian protein deacetylases homologous to the yeast master lifespan regulator Sir2. In recent years, the sirtuin protein deacetylases have emerged as candidate therapeutic targets for many human diseases, including metabolic and age-dependent neurological disorders. In non-neuronal cells, SIRT2 has been shown to function as a tubulin deacetylase and a key regulator of cell division and differentiation. However, the distribution and function of the SIRT2 microtubule (MT) deacetylase in differentiated, postmitotic neurons remain largely unknown. Here, we show abundant and preferential expression of specific isoforms of SIRT2 in the mammalian central nervous system and find that a previously uncharacterized form, SIRT2.3, exhibits age-dependent accumulation in the mouse brain and spinal cord. Further, our studies reveal that focal areas of endogenous SIRT2 expression correlate with reduced α-tubulin acetylation in primary mouse cortical neurons and suggest that the brain-enriched species of SIRT2 may function as the predominant MT deacetylases in mature neurons. Recent reports have demonstrated an association between impaired tubulin acetyltransferase activity and neurodegenerative disease; viewed in this light, our results showing age-dependent accumulation of the SIRT2 neuronal MT deacetylase in wild-type mice suggest a functional link between tubulin acetylation patterns and the aging brain.

The Sirtuin 2 Inhibitor AK-7 Is Neuroprotective in Huntington’s Disease Mouse Models

Inhibition of sirtuin 2 (SIRT2) deacetylase mediates protective effects in cell and invertebrate models of Parkinsons disease and Huntingtons disease (HD). Here we report the in vivo efficacy of a brain-permeable SIRT2 inhibitor in two genetic mouse models of HD. Compound treatment resulted in improved motor function, extended survival, and reduced brain atrophy and is associated with marked reduction of aggregated mutant huntingtin, a hallmark of HD pathology. Our results provide preclinical validation of SIRT2 inhibition as a potential therapeutic target for HD and support the further development of SIRT2 inhibitors for testing in humans.

A mitochondrial targeted fusion peptide exhibits remarkable cytotoxicity

A potent cytotoxic peptide (r7-kla) was synthesized by incorporating a mitochondrial membrane disrupting peptide, kla (klaklakklaklak), with a cell-penetrating domain, r7 (rrrrrrr). The IC50 of r7-kla (3.54 ± 0.11 μmol/L) was more than two orders of magnitude lower than that of kla. r7-kla induced cell death in both in vitro and in vivo environments, and showed rapid kinetics. Within minutes, the morphologic changes in cells and mitochondrial leakage were apparent by microscopy and was consistent with rapid apoptosis. Our results suggested that r7-kla is an apoptosis inducer and can be potentially used as an antitumor agent, especially when combined with the appropriate systemic delivery systems. [Mol Cancer Ther 2006;5(8):1944–9]

The Sirtuin-2 Inhibitor AK7 Is Neuroprotective in Models of Parkinson's Disease but Not Amyotrophic Lateral Sclerosis and Cerebral Ischemia

Sirtuin deacetylases regulate diverse cellular pathways and influence disease processes. Our previous studies identified the brain-enriched sirtuin-2 (SIRT2) deacetylase as a potential drug target to counteract neurodegeneration. In the present study, we characterize SIRT2 inhibition activity of the brain-permeable compound AK7 and examine the efficacy of this small molecule in models of Parkinson’s disease, amyotrophic lateral sclerosis and cerebral ischemia. Our results demonstrate that AK7 is neuroprotective in models of Parkinson’s disease; it ameliorates alpha-synuclein toxicity in vitro and prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine depletion and dopaminergic neuron loss in vivo. The compound does not show beneficial effects in mouse models of amyotrophic lateral sclerosis and cerebral ischemia. These findings underscore the specificity of protective effects observed here in models of Parkinson’s disease, and previously in Huntington’s disease, and support the development of SIRT2 inhibitors as potential therapeutics for the two neurodegenerative diseases.

Non-invasive optical detection of cathepsin K-mediated fluorescence reveals osteoclast activity in vitro and in vivo

Osteoclasts degrade bone matrix by demineralization followed by degradation of type I collagen through secretion of the cysteine protease, cathepsin K. Current imaging modalities are insufficient for sensitive observation of osteoclast activity, and in vivo live imaging of osteoclast resorption of bone has yet to be demonstrated. Here, we describe a near-infrared fluorescence reporter probe whose activation by cathepsin K is shown in live osteoclast cells and in mouse models of development and osteoclast upregulation. Cathepsin K probe activity was monitored in live osteoclast cultures and correlates with cathepsin K gene expression. In ovariectomized mice, cathepsin K probe upregulation precedes detection of bone loss by micro-computed tomography. These results are the first to demonstrate non-invasive visualization of bone degrading enzymes in models of accelerated bone loss, and may provide a means for early diagnosis of upregulated resorption and rapid feedback on efficacy of treatment protocols prior to significant loss of bone in the patient.

Evaluation of Histone Deacetylases as Drug Targets in Huntington's Disease models Study of HDACs in brain tissues from R6/2 and CAG140 knock-in HD mouse models and human patients and in a neuronal HD cell model.

The family of histone deacetylases (HDACs) has recently emerged as important drug targets for treatment of slow progressive neurodegenerative disorders, including Huntington’s disease (HD). Broad pharmaceutical inhibition of HDACs has shown neuroprotective effects in various HD models. Here we examined the susceptibility of HDAC targets for drug treatment in affected brain areas during HD progression. We observed increased HDAC1 and decreased HDAC4, 5 and 6 levels, correlating with disease progression, in cortices and striata of HD R6/2 mice. However, there were no significant changes in HDAC protein levels, assessed in an age-dependent manner, in HD knock-in CAG140 mice and we did not observe significant changes in HDAC1 levels in human HD brains. We further assessed acetylation levels of α-tubulin, as a biomarker of HDAC6 activity, and found it unchanged in cortices from R6/2, knock-in, and human subjects at all disease stages. Inhibition of deacetylase activities was identical in cortical extracts from R6/2 and wild-type mice treated with a class II-selective HDAC inhibitor. Lastly, treatment with class I- and II-selective HDAC inhibitors showed similar responses in HD and wild-type rat striatal cells. In conclusion, our results show that class I and class II HDAC targets are present and accessible for chronic drug treatment during HD progression and provide impetus for therapeutic development of brain-permeable class- or isoform-selective inhibitors.

3D culture models of Alzheimer’s disease: a road map to a “cure-in-a-dish”

Alzheimer’s disease (AD) transgenic mice have been used as a standard AD model for basic mechanistic studies and drug discovery. These mouse models showed symbolic AD pathologies including β-amyloid (Aβ) plaques, gliosis and memory deficits but failed to fully recapitulate AD pathogenic cascades including robust phospho tau (p-tau) accumulation, clear neurofibrillary tangles (NFTs) and neurodegeneration, solely driven by familial AD (FAD) mutation(s). Recent advances in human stem cell and three-dimensional (3D) culture technologies made it possible to generate novel 3D neural cell culture models that recapitulate AD pathologies including robust Aβ deposition and Aβ-driven NFT-like tau pathology. These new 3D human cell culture models of AD hold a promise for a novel platform that can be used for mechanism studies in human brain-like environment and high-throughput drug screening (HTS). In this review, we will summarize the current progress in recapitulating AD pathogenic cascades in human neural cell culture models using AD patient-derived induced pluripotent stem cells (iPSCs) or genetically modified human stem cell lines. We will also explain how new 3D culture technologies were applied to accelerate Aβ and p-tau pathologies in human neural cell cultures, as compared the standard two-dimensional (2D) culture conditions. Finally, we will discuss a potential impact of the human 3D human neural cell culture models on the AD drug-development process. These revolutionary 3D culture models of AD will contribute to accelerate the discovery of novel AD drugs.

Development and characterization of 3-(benzylsulfonamido)benzamides as potent and selective SIRT2 inhibitors.

Inhibitors of sirtuin-2 deacetylase (SIRT2) have been shown to be protective in various models of Huntingtons disease (HD) by decreasing polyglutamine aggregation, a hallmark of HD pathology. The present study was directed at optimizing the potency of SIRT2 inhibitors containing the neuroprotective sulfobenzoic acid scaffold and improving their pharmacology. To achieve that goal, 176 analogues were designed, synthesized, and tested in deacetylation assays against the activities of major human sirtuins SIRT1-3. This screen yielded 15 compounds with enhanced potency for SIRT2 inhibition and 11 compounds having SIRT2 inhibition equal to reference compound AK-1. The newly synthesized compounds also demonstrated higher SIRT2 selectivity over SIRT1 and SIRT3. These candidates were subjected to a dose-response bioactivity assay, measuring an increase in α-tubulin K40 acetylation in two neuronal cell lines, which yielded five compounds bioactive in both cell lines and eight compounds bioactive in at least one of the cell lines tested. These bioactive compounds were subsequently tested in a tertiary polyglutamine aggregation assay, which identified five inhibitors. ADME properties of the bioactive SIRT2 inhibitors were assessed, which revealed a significant improvement of the pharmacological properties of the new entities, reaching closer to the goal of a clinically-viable candidate.

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients

Significance Chronic neuroinflammation and oxidative stress are likely complicit in driving disease progression in Huntingtons disease (HD). Here, we describe the mechanism of action of a unique chemical scaffold that is highly selective for activation of NRF2, the master transcriptional regulator of cellular antiinflammatory and antioxidant defense genes. The use of this scaffold revealed that NRF2 activation responses were muted in HD patient-derived neural stem cells, suggesting increased susceptibility of this critical renewable cell population to oxidative stress in HD brain. However, pharmacological activation of NRF2 was able to repress inflammatory responses in mouse microglia and astrocytes, the principal cellular mediators of neuroinflammation, and in blood monocytes from HD patients. Our results suggest multiple protective benefits of NRF2 activation for HD patients. The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntingtons disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.