Diego Piedrahita

Silencing of CDK5 Reduces Neurofibrillary Tangles in Transgenic Alzheimer's Mice

Alzheimers disease is a major cause of dementia for which treatments remain unsatisfactory. Cyclin-dependent kinase 5 (CDK5) is a relevant kinase that has been hypothesized to contribute to the tau pathology. Several classes of chemical inhibitors for CDK5 have been developed, but they generally lack the specificity to distinguish among various ATP-dependent kinases. Therefore, the efficacy of these compounds when tested in animal models cannot definitively be attributed to an effect on CDK5. However, RNA interference (RNAi) targeting of CDK5 is specific and can be used to validate CDK5 as a possible treatment target. We delivered a CDK5 RNAi by lentiviral or adenoassociated viral vectors and analyzed the results in vitro and in vivo. Silencing of CDK5 reduces the phosphorylation of tau in primary neuronal cultures and in the brain of wild-type C57BL/6 mice. Furthermore, the knockdown of CDK5 strongly decreased the number of neurofibrillary tangles in the hippocampi of triple-transgenic mice (3×Tg-AD mice). Our data suggest that this downregulation may be attributable to the reduction of the CDK5 availability in the tissue, without affecting the CDK5 kinase activity. In summary, our findings validate CDK5 as a reasonable therapeutic target for ameliorating tau pathology.

Silencing of CDK5 as potential therapy for Alzheimer ' s disease

Abstract Neurodegeneration is one of the greatest public health challenges for the 21st century. Among neurodegenerative diseases, Alzheimer’s disease (AD) is the most prevalent and best characterized. Nevertheless, despite the large investment in AD research, currently there is no effective therapeutic option. In the present review, we highlight a novel alternative, which takes advantage of the biotechnological outbreak deployed by the discovery of the RNA interference-based gene silencing mechanism, and its application as a tool for neurodegeneration treatment. Here, we highlight cyclin-dependent kinase 5 (CDK5) as a key candidate target for therapeutic gene silencing. Unlike other members of the cyclin-dependent kinase family, CDK5 does not seem to play a crucial role in cell cycle regulation. By contrast, CDK5 participates in multiple functions during nervous system development and has been established as a key mediator of Tau hyperphosphorylation and neurofibrillary pathology, thus serving as an optimal candidate for targeted therapy in the adult nervous system. We propose that the use of RNA interference for CDK5 silencing presents an attractive and specific therapeutic alternative for AD and perhaps against other tauopathies.

p35 and Rac1 underlie the neuroprotection and cognitive improvement induced by CDK5 silencing

CDK5 plays an important role in neurotransmission and synaptic plasticity in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. In a previous study, we demonstrated that RNAi knock down of CDK5 reduced the formation of neurofibrillary tangles (NFT) and prevented neuronal loss in triple transgenic Alzheimers mice. Here, we report that CDK5 RNAi protected against glutamate‐mediated excitotoxicity using primary hippocampal neurons transduced with adeno‐associated virus 2.5 viral vector eGFP‐tagged scrambled or CDK5 shRNA‐miR during 12 days. Protection was dependent on a concomitant increase in p35 and was reversed using p35 RNAi, which affected the down‐stream Rho GTPase activity. Furthermore, p35 over‐expression and constitutively active Rac1 mimicked CDK5 silencing‐induced neuroprotection. In addition, 3xTg‐Alzheimers disease mice (24 months old) were injected in the hippocampus with scrambled or CDK5 shRNA‐miR, and spatial learning and memory were performed 3 weeks post‐injection using ‘Morris’ water maze test. Our data showed that CDK5 knock down induced an increase in p35 protein levels and Rac activity in triple transgenic Alzheimers mice, which correlated with the recovery of cognitive function; these findings confirm that increased p35 and active Rac are involved in neuroprotection. In summary, our data suggest that p35 acts as a mediator of Rho GTPase activity and contributes to the neuroprotection induced by CDK5 RNAi. CDK5 plays an important role in neurotransmission in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. Our findings suggest that p35/Rac1 signaling is critical in the CDK5 shRNAmiR‐induced neuroprotection against glutamate neurotoxicity and is also correlated with the recovery of cognitive function in 3xTg‐AD mice. CDK5 shRNAmiR blocks calpain activation, and the cleavage of p35 to p25 produced by glutamate, which generates neuroprotection in a p35 up‐regulation dependent mode and its down‐stream control of Rho GTPases, such as Rac1 and RhoA.

β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

β-site APP cleaving enzyme 1 (BACE1) initiates APP cleavage, which has been reported to be an inducer of tau pathology by altering proteasome functions in Alzheimer’s disease (AD). However, the exact relationship between BACE1 and PHF (Paired Helical Filaments) formation is not clear. In this study, we confirm that BACE1 and Hsc70 are upregulated in the brains of AD patients, and we demonstrate that both proteins show enhanced expression in lipid rafts from AD-affected triple transgenic mouse brains. BACE1 targeting increased Hsc70 levels in the membrane and cytoplasm fractions and downregulated Hsp90 and CHIP in the nucleus in the hippocampi of 3xTg-AD mice. However, these observations occurred in a proteasome-independent manner in vitro. The BACE1miR-induced reduction of soluble hyperphosphorylated tau was associated with a decrease in MAPK activity. However, the BACE1 RNAi-mediated reduction of hyperphosphorylated tau was only blocked by 3-MA (3-methyladenine) in vitro, and it resulted in the increase of Hsc70 and LAMP2 in lipid rafts from hippocampi of 3xTg-AD mice, and upregulation of survival and homeostasis signaling. In summary, our findings suggest that BACE1 silencing neuroprotects reducing soluble hyperphosphorylated tau, modulating certain autophagy-related proteins in aged 3xTg-AD mice.

BACE1 RNAi Restores the Composition of Phosphatidylethanolamine-Derivates Related to Memory Improvement in Aged 3xTg-AD Mice.

β-amyloid (Aβ) is produced by the β-secretase 1 (BACE1)-mediated enzymatic cleavage of the amyloid precursor protein through the amyloidogenic pathway, making BACE1 a therapeutic target against Alzheimer’s disease (AD). Alterations in lipid metabolism are a risk factor for AD by an unknown mechanism. The objective of this study was to determine the effect of RNA interference against BACE1 (shBACEmiR) on the phospholipid profile in hippocampal CA1 area in aged 3xTg-AD mice after 6 and 12 months of treatment compared to aged PS1KI mice. The shBACEmiR treatment induced cognitive function recovery and restored mainly the fatty acid composition of lysophosphatidylethanolamine and etherphosphatidylethanolamine, reduced the cPLA2’s phosphorylation, down-regulated the levels of arachidonic acid and COX2 in the hippocampi of 3xTg-AD mice. Together, our findings suggest, for the first time, that BACE1 silencing restores phospholipids composition which could favor the recovery of cellular homeostasis and cognitive function in the hippocampus of triple transgenic AD mice.

Molecular characterization of Marek's disease virus in a poultry layer farm from Colombia

&NA; Mareks disease (MD) is a lymphoproliferative disease caused by an Alphaherpesvirus, genus Mardivirus, serotype 1 (Gallid Herpesvirus 2, GaHV‐2) that includes all known pathogenic strains. In addition to Mareks disease virus (MDV) serotype 1, the genus includes 2 distinct nonpathogenic serotypes: serotype 2 (GaHV‐3) and serotype 3 (Meleagridis Herpesvirus 1, MeHV‐1) which are used in commercially available vaccines against MD. As a result of vaccination, clinical signs are not commonly observed, and new cases are usually associated with emerging variant strains against which the vaccines are less effective. In this study, a commercial layer farm showing clinical signs compatible with MDV infection was evaluated. Histological lesions and positive immunohistochemistry in the sciatic nerve and thymus were compatible with cytolytic phase of MD. GaHV‐2, GaHV‐3 and MeHV‐1 were identified by PCR and qPCR in blood samples from 17 birds with suspected MD. Analysis of the Meq gene of the Colombian GaHV‐2 isolate revealed a 99% sequence identity with Asian strains, and in the phylogenetic analysis clustered with vv+ MDV. The analysis of amino acid alignments demonstrated an interruption of the proline rich region in P176A, P217A and P233L positions, which are generally associated with vv+ strains. Some of these changes, such as P233L and L258S positions have not been reported previously. In addition, primary cell cultures inoculated with lymphocytes isolated from the spleen showed typical cytopathic effect of GaHV‐2 at 5 d post infection. Based on the molecular analysis, the results from this study indicate the presence of vv+ MDV infection in commercial birds for the first time in Colombia. It is recommended to perform further assays in order to demonstrate the pathotype characteristics in vivo.

A preliminary survey of Trichinella spp. in pigs raised under controlled housing conditions in Colombia: 2014–2016

A preliminary survey of Trichinella spp. infection was conducted in Colombian swine herds between 2014 and 2016. A total of 1,773 pigs reared on farms under controlled housing conditions and processed in 34 slaughterhouses were tested either by the artificial digestion of pooled muscle samples (n = 1,173) or by serology (n = 600). In addition, 550 rats trapped on 29 swine farm premises were also tested by artificial digestion. No positive pig samples were detected. Similarly, no Trichinella spp. muscle larvae were detected in rats. These results are in agreement with the lack of historical Trichinella infection reports in domestic and wild animals and humans in Colombia. However, a more extensive epidemiological investigation and a continuous surveillance program are needed to continue declaring swine herds in Colombia free of Trichinella infection.