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Mechanisms of tissue damage in Huntington's disease involve excitotoxicity, mitochondrial damage, and inflammation, including microglia activation. Immunomodulatory and anti-protein aggregation properties of tetracyclines were demonstrated in several disease models. In the present study, the neuroprotective and anti-inflammatory effects of the tetracycline doxycycline were investigated in the mouse model of HD disease R6/2. Transgenic mice were daily treated with doxycycline 20 mg/kg, starting from 4 weeks of age. After sacrifice, histological and immunohistochemical studies were performed. We found that doxycycline-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the saline-treated ones. Primary outcome measures such as striatal atrophy, neuronal intranuclear inclusions, and the negative modulation of microglial reaction revealed a neuroprotective effect of the compound. Doxycycline provided a significantly increase of activated CREB and BDNF in the striatal neurons, along with a down modulation of neuroinflammation, which, combined, might explain the beneficial effects observed in this model. Our findings show that doxycycline treatment could be considered as a valid therapeutic approach for HD.

Inflammation significantly impacts the progression of Huntington's disease (HD) and the mutant HTT protein determines a pro-inflammatory activation of microglia. Mesenchymal stem/stromal cells (MSC) from the amniotic membrane (hAMSC), and their conditioned medium (CM-hAMSC), have been shown to possess protective effects in vitro and in vivo in animal models of immune-based disorders and of traumatic brain injury, which have been shown to be mediated by their immunomodulatory properties. In this study, in the R6/2 mouse model for HD we demonstrate that mice treated with CM-hAMSC display less severe signs of neurological dysfunction than saline-treated ones. CM-hAMSC treatment significantly delayed the development of the hind paw clasping response during tail suspension, reduced deficits in rotarod performance, and decreased locomotor activity in an open field test. The effects of CM-hAMSC on neurological function were reflected in a significant amelioration in brain pathology, including reduction in striatal atrophy and the formation of striatal neuronal intranuclear inclusions. In addition, while no significant increase was found in the expression of BDNF levels after CM-hAMSC treatment, a significant decrease of microglia activation and inducible nitric oxide synthase levels were observed. These results support the concept that CM-hAMSC could act by modulating inflammatory cells, and more specifically microglia., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Neuroglobin (Ngb) is expressed in the central and peripheral nervous system, cerebrospinal fluid, retina, and endocrine tissues where it is involved in binding O 2 and other gasotransmitters. Several studies have highlighted its endogenous neuroprotective function. Huntington's disease (HD), a dominant hereditary disease, is characterized by the gradual loss of neurons in discrete areas of the central nervous system. We analyzed the expression of Ngb in the brain tissue of a mouse model of HD, in order to define the role of Ngb with respect to individual cell type vulnerability in HD and to gender and age of mice. Our results showed different expressions of Ngb among neurons of a specific region and between different brain regions. We evidenced a decreased intensity of Ngb at 13 weeks of age, compared to 7 weeks of age. The double immunofluorescence and fluorescence resonance energy transfer (FRET) experiments showed that the co-localization between Ngb and huntingtin at the subcellular level was not close enough to account for a direct interaction. We also observed a different expression of Ngb in the striatum, depending on the sex and age of animals. These findings provide the first experimental evidence for an adaptive response of Ngb in HD, suggesting that Ngb may exert neuroprotective effects in HD beyond its role in reducing sensitivity to oxidative stress.

Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in physiological processes as DNA repair, genomic stability, and apoptosis. Moreover, published studies demonstrated that PARP-1 mediates necrotic cell death in response to excessive DNA damage under certain pathological conditions. In Huntington's disease brains, PARP immunoreactivity was described in neurons and in glial cells, thereby suggesting the involvement of apoptosis in HD. In this study, we sought to determine if the PARP-1 inhibitor exerts a neuroprotective effect in R6/2 mutant mice, which recapitulates, in many aspects, human HD. Transgenic mice were treated with the PARP-1 inhibitor INO-1001 mg/Kg daily starting from 4 weeks of age. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that INO 1001-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as striatal atrophy, morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. INO-1001 was effective in significantly increasing activated CREB and BDNF in the striatal spiny neurons, which might account for the beneficial effects observed in this model. Our findings show that PARP-1 inhibition could be considered as a valid therapeutic approach for HD.

Huntington's disease (HD) is an autosomal-dominant inherited neurodegenerative disorder characterized by motor dysfunction, cognitive decline, and emotional and psychiatric disturbances. The genetic mutation is characterized by a CAG expansion, resulting in the formation of a mutant huntingtin protein with an expanded polyglutamine repeat region. Mutated huntingtin has been shown to impair a number of physiological activities by interacting with several factors. In particular, cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) are severely affected by mutant huntingtin. In this view, drugs targeted at counteracting CREB loss of function and BDNF decrease have been considered as powerful tools to treat HD. Recently, cyclic nucleotide phosphodiesterase (PDE) inhibitors have been used successfully to increase levels of CREB and BDNF in HD models. Indeed, PDE4, 5 or 10 inhibitors have been shown to afford neuroprotection and modulation of CREB and BDNF. In this review, we will summarize the data supporting the use of PDE inhibitors as the therapeutical approach to fight HD and we will discuss the possible mechanisms of action underlying these effects.

Loss of huntingtin-mediated BDNF gene transcription has been shown to occur in HD and thus contribute to the degeneration of the striatum. Several studies have indicated that an increase in BDNF levels is associated with neuroprotection and amelioration of neurological signs in animal models of HD. In a recent study, an increase in BDNF mRNA and protein levels was recorded in mice administered recombinant BDNF peripherally. Chronic, indwelling osmotic mini-pumps containing either recombinant BDNF or saline were surgically placed in R6/2 or wild-type mice from 4 weeks of age until euthanasia. Neurological evaluation (paw clasping, rotarod performance, locomotor activity in an open field) was performed. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that BDNF- treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as brain volume, striatal atrophy, size and morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. BDNF was effective in increasing significantly the levels of activated CREB and of BDNF the striatal spiny neurons. Moreover, systemically administered BDNF increased the synthesis of BDNF as demonstrated by RT-PCR, and this might account for the beneficial effects observed in this model.

In Huntington's disease (HD) mutant huntingtin protein impairs the function of several transcription factors, in particular the cAMP response element-binding protein (CREB). CREB activation can be increased by targeting phosphodiesterases such as phospohodiesterase 4 (PDE4) and phosphodiesterase 10A (PDE10A). Indeed, both PDE4 inhibition (DeMarch et al., 2008) and PDE10A inhibition (Giampà et al., 2010) proved beneficial in the R6/2 mouse model of HD. However, Hebb et al. (2004) reported PDE10A decline in R6/2 mice. These findings raise the issue of how PDE10A inhibition is beneficial in HD if such enzyme is lost. R6/2 mice and their wild type littermates were treated with the PDE10A inhibitor TP10 (a gift from Pfizer) or saline, sacrificed at 5, 9, and 13 weeks of age, and single and double label immunohistochemistry and western blotting were performed. PDE10A increased dramatically in the spiny neurons of R6/2 compared to the wild type mice. Conversely, in the striatal cholinergic interneurons, PDE10A was lower and it did not change significantly with disease progression. In the other subsets of striatal interneurons (namely, parvalbuminergic, somatostatinergic, and calretininergic interneurons) PDE10A immunoreactivity was higher in the R6/2 compared to the wild-type mice. In the TP10 treated R6/2, PDE10A levels were lower than in the saline treated mice in the medium spiny neurons, whereas they were higher in all subsets of striatal interneurons except for the cholinergic ones. However, in the whole striatum densitometry studies, PDE10A immunoreactivity was lower in the R6/2 compared to the wild-type mice. Our study demonstrates that PDE10A is increased in the spiny neurons of R6/2 mice striatum. Thus, the accumulation of PDE10A in the striatal projection neurons, by hydrolyzing greater amounts of cyclic nucleotides, is likely to contribute to cell damage in HD. Consequently, the beneficial effect of TP10 in HD models (Giampà et al., 2009, 2010) is explained by the efficiency of such compound in counteracting this phenomenon and therefore increasing the availability of cyclic nucleotides., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The mitogen-activated protein kinases (MAPKs) superfamily comprises three major signaling pathways: the extracellular signal-regulated protein kinases (ERKs), the c-Jun N-terminal kinases or stress-activated protein kinases (JNKs/SAPKs) and the p38 family of kinases. ERK 1/2 signaling has been implicated in a number of neurodegenerative disorders, including Huntington's disease (HD). Phosphorylation patterns of ERK 1/2 and JNK are altered in cell models of HD. In this study, we aimed at studying the correlations between ERK 1/2 and the neuronal vulnerability to HD degeneration in the R6/2 transgenic mouse model of HD. Single and double-label immunofluorescence for phospho-ERK (pERK, the activated form of ERK) and for each of the striatal neuronal markers were employed on perfusion-fixed brain sections from R6/2 and wild-type mice. Moreover, Phosphodiesterase 4 inhibition through rolipram was used to study the effects on pERK expression in the different types of striatal neurons. We completed our study with western blot analysis. Our study shows that pERK levels increase with age in the medium spiny striatal neurons and in the parvalbumin interneurons, and that rolipram counteracts such increase in pERK. Conversely, cholinergic and somatostatinergic interneurons of the striatum contain higher levels of pERK in the R6/2 mice compared to the controls. Rolipram induces an increase in pERK expression in these interneurons. Thus, our study confirms and extends the concept that the expression of phosphorylated ERK 1/2 is related to neuronal vulnerability and is implicated in the pathophysiology of cell death in HD., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The receptor for advanced glycation end (RAGE) products is a multi-ligand receptor that belongs to the immunoglobulin superfamily of cell surface receptors, whose ligands are known to be upregulated in neuropathological conditions. RAGE upregulation has been described in neurodegenerative diseases, such as Alzheimer's disease, Creutzfeldt-Jakob's disease and Huntington's disease (HD). To analyze in detail the implication of RAGE in HD, we studied the immunohistochemical distribution of RAGE in the striatum of the R6/2 mouse model of HD, with particular attention to the neuronal subpopulations and their relative vulnerability to HD neurodegeneration. We show that RAGE immunoreactivity is evenly distributed to the cytoplasm of neurons in the wild type mouse, while it is finely granular in the cytoplasm of striatal neurons of R6/2 mouse. RAGE is distributed in 98% of spiny projection neurons, both in the normal mouse and in the R6/2. RAGE co-localizes with all of the striatal interneuron subsets both in the wild-type and in the R6/2 mouse. However, the intensity of RAGE immunoreactivity is significantly higher in the spiny neurons and in the PARV neurons of R6/2 mouse, whereas it is comparable between R6/2 and wild-type in the cholinergic and somatostatinergic interneurons. These data support the concept that RAGE is upregulated in the neurodegenerative process of HD, and suggests that its activation is related to the individual vulnerability of the striatal neuronal subtype., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Huntington's disease is a devastating neurodegenerative condition for which there is no therapy to slow disease progression. The particular vulnerability of striatal medium spiny neurons to Huntington's pathology is hypothesized to result from transcriptional dysregulation within the cAMP and CREB signaling cascades in these neurons. To test this hypothesis, and a potential therapeutic approach, we investigated whether inhibition of the striatal-specific cyclic nucleotide phosphodiesterase PDE10A would alleviate neurological deficits and brain pathology in a highly utilized model system, the R6/2 mouse., Methodology/principal Findings: R6/2 mice were treated with the highly selective PDE10A inhibitor TP-10 from 4 weeks of age until euthanasia. TP-10 treatment significantly reduced and delayed the development of the hind paw clasping response during tail suspension, deficits in rotarod performance, and decrease in locomotor activity in an open field. Treatment prolonged time to loss of righting reflex. These effects of PDE10A inhibition on neurological function were reflected in a significant amelioration in brain pathology, including reduction in striatal and cortical cell loss, the formation of striatal neuronal intranuclear inclusions, and the degree of microglial activation that occurs in response to the mutant huntingtin-induced brain damage. Striatal and cortical levels of phosphorylated CREB and BDNF were significantly elevated., Conclusions/significance: Our findings provide experimental support for targeting the cAMP and CREB signaling pathways and more broadly transcriptional dysregulation as a therapeutic approach to Huntington's disease. It is noteworthy that PDE10A inhibition in the R6/2 mice reduces striatal pathology, consistent with the localization of the enzyme in medium spiny neurons, and also cortical pathology and the formation of neuronal nuclear inclusions. These latter findings suggest that striatal pathology may be a primary driver of these secondary pathological events. More significantly, our studies point directly to an accessible new therapeutic approach to slow Huntington's disease progression, namely, PDE10A inhibition. There is considerable activity throughout the pharmaceutical industry to develop PDE10A inhibitors for the treatment of basal ganglia disorders. The present results strongly support the investigation of PDE10A inhibitors as a much needed new treatment approach to Huntington's disease.

Decreased activity of cAMP responsive element-binding protein (CREB) is thought to contribute to the death of striatal medium spiny neurons in Huntington's disease (HD). Therefore, therapies that increase levels of activated CREB, may be effective in fighting neurodegeneration in HD. In this study, we sought to determine whether the phosphodiesterase type 10 (PDE10A) inhibitor TP10 exerts a neuroprotective effect in an excitotoxic model of HD. Rats were surgically administered with quinolinic acid into striatum and subsequently treated with TP10 daily for two or eight weeks. After 2 weeks of TP10 treatment, striatal lesion size was 52% smaller and the surviving cell number was several times higher than in the vehicle-treated group. These beneficial effects of TP10 were maintained through 8 weeks. TP10 treatment also increased significantly the levels of activated CREB in the striatal spiny neurons, which is hypothesized to be a contributing mechanism for the neuroprotective effect. Our findings suggest PDE10A inhibition as a novel neuroprotective approach to the treatment of HD and confirm the importance of phosphodiesterase inhibition in fighting the disease.

A possible neuroprotective role has been recently suggested for 3H3MGCoA reductase inhibitors. Here, we sought to determine whether simvastatin exerts a neuroprotective effect in our rat model of HD. Rats were surgically administered quinolinic acid and treated with simvastatin 1mg/kg intraperitoneally (i.p.) once daily up to 2 or 8 weeks. Two more groups of animals received a pretreatment with 1mg/kg simvastatin i.p. for 2 weeks before the QA lesion and then were treated with simvastatin for the following 2 weeks or 8 weeks, respectively. In the simvastatin treated groups (both pretreated and non-pretreated), striatal lesion size was about 36% smaller while neuronal counts where higher than in the vehicle treated ones at 2 weeks. The neuroprotective effects of simvastatin was still evident at 8 weeks post lesion, where the non-pretreated group had a 8% smaller lesion size than the saline group, and the pretreated group had an 11% smaller lesion size than the saline group. Simvastatin also induced immunoreactivity for Bcl-2, an anti-apoptotic factor, on one hand, and down-regulated immunoreactivity for Bax, a proapoptotic factor. Bcl-2/Bax modulation can account, at least partly, for the beneficial effect of simvastatin in our rodent model of striatal degeneration. Our findings show that statins could be explored as possible neuroprotective agents for neurodegenerative disorders such as HD.

We have previously showed that rolipram, a phosphodiesterase type IV inhibitor, displays a neuroprotective effect in a rat quinolinic acid model of HD [DeMarch Z., Giampa C., Patassini S., Martorana A., Bernardi G. and Fusco F.R., (2007) Beneficial effects of rolipram in a quinolinic acid model of striatal excitotoxicity. Neurobiol. Dis. 25:266-273.]. In this study, we sought to determine if rolipram exerts a neuroprotective effect in R6/2 mutant mice, which recapitulates, in many aspects, human HD [Mangiarini L., Sathasivam K., Seller M., Cozens B., Harper A., Hetherington C., Lawton M., Trottier Y., Lehrach H., Davies S.W. and Bates G.P. (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell. 87:493-506]. Transgenic mice were treated with rolipram 1.5 mg/kg daily starting from 4 weeks of age. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that rolipram-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as brain volume, striatal atrophy, size and morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. Rolipram was effective in increasing significantly the levels of activated CREB and of BDNF the striatal spiny neurons, which might account for the beneficial effects observed in this model. Our findings show that rolipram could be considered as a valid therapeutic approach for HD.

Activity of c-AMP responsive element-binding protein (CREB) is decreased in Huntington's disease (HD). Such decrease was also described by our group in the quinolinic acid lesion model of striatal excitotoxicity. The phosphodiesterase type IV inhibitor rolipram increases CREB phosphorylation. Such drug has a protective effect in global ischaemia and embolism in rats. In this study, we sought to determine whether rolipram displays a neuroprotective effect in our rat model of HD. Animals were surgically administered QA and subsequently treated with rolipram daily up to 2 and 8 weeks respectively. After these time points, rats were sacrificed and immunohistochemical studies were performed in the striata. In the rolipram-treated animals, striatal lesion size was about 62% smaller that in the vehicle-treated ones at 2 weeks time point. Moreover, the surviving cell number was several times higher in the rolipram-treated animals than in the vehicle group at both time points. Rolipram also showed to be effective in increasing significantly the levels of activated CREB in the striatal spiny neurons, which accounts mostly for its beneficial effect in our rodent model of excitotoxicity. Our findings show that rolipram could be considered as a valid therapeutic approach for HD.

Recent evidence has shown that the activity of cAMP responsive element-binding protein (CREB) and of CREB-binding protein (CBP) is decreased in Huntington's disease (HD) [Steffan et al. (2000)Proc. Natl Acad. Sci. USA, 97, 6763-6768; Gines et al. (2003)Hum. Mol. Genet., 12, 497-508; Rouaux et al. (2004) Biochem. Pharmacol., 68, 1157-1164; Sugars et al. (2004)J. Biol. Chem., 279, 4988-4999]. Such decrease is thought to reflect the impaired energy metabolism observed in a HD mouse model, where a decline in striatum cAMP levels has been observed [Gines et al. (2003)Hum. Mol. Genet., 12, 497-508]. Increased levels of CREB have also been demonstrated to exert neuroprotective functions [Lonze & Ginty (2002)Neuron, 35, 605-623; Lonze et al. (2002)Neuron, 34, 371-385]. Our study aimed to investigate the distribution of CREB in the neuronal subpopulations of the striatum in normal rats compared to the HD model of quinolinic acid lesion. Twenty-five Wistar rats were administered quinolinic acid 100 mm into the right striatum, and killed after 24 h, 48 h, 1 week, 2 weeks, and six weeks, respectively. The contralateral striata were used as controls. Dual-label immunofluorescence was employed using antibodies against phosphorylated CREB and each of the different neuronal subpopulations markers. Our results show that activated CREB levels decrease progressively in projection neurons and parvalbumin (PARV) and calretinin (CALR) interneurons, whereas such levels remain stable in cholinergic and somatostatin interneurons. Thus, we speculate that the ability of cholinergic interneurons to maintain their levels of CREB after excitotoxic lesions is one of the factors determining their protection in Huntington's disease.