Central nervous system (CNS) trauma can result in tissue disruption, neuronal and axonal degeneration, and neurological dysfunction. The limited spontaneous CNS repair in adulthood and aging is often insufficient to overcome disability. Several investigations have demonstrated that targeting HDAC activity can protect neurons and glia and improve outcomes in CNS injury and disease models. However, the enthusiasm for pan-HDAC inhibition in treating neurological conditions is tempered by their toxicity toward a host of CNS cell types -a biological extension of their anticancer properties. Identification of the HDAC isoform, or isoforms, that specifically mediate the beneficial effects of pan-HDAC inhibition could overcome this concern. Here, we show that pan-HDAC inhibition not only promotes neuronal protection against oxidative stress, a common mediator of injury in many neurological conditions, but also promotes neurite growth on myelin-associated glycoprotein and chondroitin sulfate proteoglycan substrates. Real-time PCR revealed a robust and selective increase in HDAC6 expression due to injury in neurons. Accordingly, we have used pharmacological and genetic approaches to demonstrate that inhibition of HDAC6 can promote survival and regeneration of neurons. Consistent with a cytoplasmic localization, the biological effects of HDAC6 inhibition appear transcriptionindependent. Notably, we find that selective inhibition of HDAC6 avoids cell death associated with pan-HDAC inhibition. Together, these findings define HDAC6 as a potential nontoxic therapeutic target for ameliorating CNS injury characterized by oxidative stressinduced neurodegeneration and insufficient axonal regeneration.HDAC inhibitor ͉ histone deacetylase ͉ neuroprotection ͉

is sufficient for neuroprotection, it is not necessary for HDAC inhibitor neuroprotection, because these agents can completely protect neurons cultured from p21 waf1/cip1 -null mice. Together these findings demonstrate (1) that pulse inhibition of HDACs in cortical neurons can induce neuroprotection without apparent toxicity; (2) that p21 waf1/cip1 is sufficient but not necessary to mimic the protective effects of HDAC inhibition; and (3) that oxidative stress in this model induces neuronal cell death via cell cycle-independent pathways that can be inhibited by a cytosolic, noncanonical action of p21 waf1/cip1 .

We compare the ability of two structurally different classes of epigenetic modulators, namely, histone deacetylase (HDAC) inhibitors containing either a hydroxamate or a mercaptoacetamide as the zinc binding group, to protect cortical neurons in culture from oxidative stress-induced death. This study reveals that some of the mercaptoacetamide-based HDAC inhibitors are fully protective, whereas the hydroxamates show toxicity at higher concentrations. Our present results appear to be consistent with the possibility that the mercaptoacetamide-based HDAC inhibitors interact with a different subset of the HDAC isozymes [less activity at HDAC1 and 2 correlates with less inhibitor toxicity], or alternatively, are interacting selectively with only the cytoplasmic HDACs that are crucial for protection from oxidative stress.

We compare three structurally different classes of histone deacetylase (HDAC) inhibitors that contain benzamide, hydroxamate, or thiol groups as the zinc binding group (ZBG) for their ability to protect cortical neurons in culture from cell death induced by oxidative stress. This study reveals that none of the benzamide-based HDAC inhibitors (HDACIs) provides any neuroprotection whatsoever, in distinct contrast to HDACIs that contain other ZBGs. Some of the sulfur-containing HDACIs, namely the thiols, thioesters, and disulfides present modest neuroprotective activity but show toxicity at higher concentrations. Taken together, these data demonstrate that the HDAC6-selective mercaptoacetamides that were reported previously provide the best protection in the homocysteic acid model of oxidative stress, thus further supporting their study in animal models of neurodegenerative diseases.

Alzheimer's disease (AD) is a well-studied process characterized by the presence of amyloid plaques and neurofibrillary tangles. In this study, a series of protein kinase C (PKC) activators were investigated, some of which also exhibit histone deacetylases (HDACs) inhibitory activity, under the hypothesis that such compounds might provide a new path forward in the discovery of drugs for the treatment of AD. The PKC activating properties of these drugs were expected to enhance the α-secretase pathway in the processing of amyloid precursor protein (APP), while their HDAC inhibition was anticipated to confer neuroprotective activity. We found that the benzolactams compounds 9 and 11-14 caused a concentration-dependent increase in sAPPα and decrease in β-amyloid (Aβ) production using concentrations of these drugs in the range of 0.1∼10 μM, consistent with a shift of APP metabolism towards the α-secretase-processing pathway. Moreover, 9-14 showed neuroprotective effects in the 10 to 20 μM range in the homocysteate (HCA) cortical neuron model of oxidative stress. In parallel, we found that the most neuroprotective compounds caused increased levels of histone acetylation (H4), thus indicating their likely ability to inhibit histone deacetylase activity. As the majority of the compounds studied also show nanomolar binding affinities for PKC, we conclude that it is possible to design, de novo, agents that combine both PKC activating properties along with HDAC inhibitory properties, thereby resulting in agents capable of modulating amyloid processing while showing neuroprotection. These findings may offer a new approach to therapies that exhibit disease-modifying effects, as opposed to symptomatic relief, in the treatment of AD.

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Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed Protein Kinase C substrate that has emerged as a potential therapeutic target for amelioration of mucin secretion during chronic obstructive pulmonary disease. MARCKS also plays a key role in regulation of neutrophil adhesion, migration and degranulation. Given its biological role in immune cells, we hypothesized that MARCKS may play an integral role in cytokine secretion. MARCKS protein is highly conserved between the dog and human, and importantly, the N-terminal 24 amino acids are virtually identical between species, allowing us to use the well-characterized MANS peptide to inhibit canine MARCKS function through the N-terminus. Using isolated neutrophils, we evaluated the effect of MARCKS inhibition on LPS-induced cytokine production. We found that pre-treatment of canine neutrophils with MANS peptide significantly reduced the secretion of a broad range of LPS-induced cytokines, including IL-8, CXCL1 and TNFα, in comparison to untreated cells or those treated with a scrambled control peptide. This reduction in IL-8 was maintained when MANS was administered 2 hours after LPS-treatment of cells. Reduction in IL-8 secretion was found not to be due to protein retention or cell death. Our observations identify MARCKS protein as a promising therapeutic target in treatment of inflammatory disease, particularly those syndromes attributed to neutrophil influx and inflammatory cytokine production.

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