BACKGROUND:The Huntingtin (HTT) N-terminal domains encoded by Huntingtin's (HTT) exon 1 consist of an N17 domain, the polyglutamine (polyQ) stretch and a proline-rich region (PRR). These domains are conserved in mammals and have been hypothesized to modulate HTT's functions in the developing and adult CNS, including DNA damage repair and autophagy.OBJECTIVE:This study longitudinally characterizes the in vivo consequences of deleting the murine Htt N-terminal domains encoded by Htt exon 1.METHODS:Knock-in mice with a deletion of Htt exon 1 sequences (HttΔE1) were generated and bred into the C57BL/6J congenic genetic background. Their behavior, DNA damage response, basal autophagy, and glutamatergic synapse numbers were evaluated.RESULTS:Progeny from HttΔE1/+ intercrosses are born at the expected Mendelian frequency but with a distorted male to female ratio in both the HttΔE1/ΔE1 and Htt+/+ offspring. HttΔE1/ΔE1 adults exhibit a modest deficit in accelerating rotarod performance, and an earlier increase in cortical and striatal DNA damage with elevated neuronal pan-nuclear 53bp1 levels compared to Htt+/+ mice. However, a normal response to induced DNA damage, normal levels of basal autophagy markers, and no significant differences in corticocortical, corticostriatal, thalamocortical, or thalamostriatal synapses numbers were observed compared to controls.CONCLUSION:Our results suggest that deletion of the Htt N-terminus encoded by the Htt exon 1 does not affect Htt's critical role during embryogenesis, but instead, may have a modest effect on certain motor tasks, basal levels of DNA damage in the brain, and Htt function in the testis.

Initial dysregulation of amyloid beta (Ab) results in hyperexcitation, which in turn results in generation of toxic Ab oligomers and formation of plaques. One of the main mechanisms of Ab generation is through AMPA and NMDA receptor mediated activity-induced endocytosis of amyloid precursor protein (APP). These endocytosis events bring APP into contact with enzymes that can cleave APP (i.e. b-secretase), leading to generation of Ab. Our lab has recently identified polo-like kinase 2 (Plk2) as an important modulator for homeostatic synaptic plasticity. Plk2 also modulates APP endocytosis and processing, and it can phosphorylate both APP and tau after hyperexcitation. To determine if there is an association between generation of Ab and changes in Plk2 levels in people, we performed immunohistochemical staining in post-mortem AD brain samples from regions of the brain most affected by AD. We also tested the ability of two Plk inhibitors to specifically and robustly decrease Ab generation in AD model mice. We were able to observe a robust increase in Plk2 staining in AD patient hippocampal tissue samples that was not seen in regions of the brain unaffected by AD (cerebellum and occipital lobe). Treatment with both Plk inhibitors resulted in a significant decrease in Ab levels in AD mouse brains. These experiments suggest that Plk2 may be a key regulator in promoting hyperactivity and the generation and secretion of Ab in early AD, and may therefore be a promising target for patient intervention.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by pathological hallmarks of neurofibrillary tangles and amyloid plaques. The plaques are formed by aggregation and accumulation of amyloid β (Aβ), a cleavage fragment of amyloid precursor protein (APP). Enhanced neuronal activity and seizure events are frequently observed in AD, and elevated synaptic activity promotes Aβ production. However, the mechanisms that link synaptic hyperactivity to APP processing and AD pathogenesis are not well understood. We previously found that Polo-like kinase 2 (Plk2), a homeostatic repressor of neuronal overexcitation, promotes APP β-processing in vitro. Here, we report that Plk2 stimulates Aβ production in vivo, and that Plk2 levels are elevated in a spatiotemporally regulated manner in brains of AD mouse models and human AD patients. Genetic disruption of Plk2 kinase function reduces plaque deposits and activity-dependent Aβ production. Furthermore, pharmacological Plk2 inhibition hinders Aβ formation, synapse loss, and memory decline in an AD mouse model. Thus, Plk2 links synaptic overactivity to APP β-processing, Aβ production, and disease-relevant phenotypes in vivo, suggesting that Plk2 may be a potential target for AD therapeutics.

Brain activity levels are tightly regulated to minimize imbalances in activity state. Deviations from the normal range of activity are deleterious and often associated with neurological disorders. To maintain optimal levels of activity, regulatory mechanisms termed homeostatic synaptic plasticity establish desired 'set points' for neural activity, monitor the network for deviations from the set point and initiate compensatory responses to return activity to the appropriate level that permits physiological function [1,2]. We speculate that impaired homeostatic control may contribute to the etiology of various neurological disorders including epilepsy and Alzheimer's disease, two disorders that exhibit hyperexcitability as a key feature during pathogenesis. Here, we will focus on recent progress in developing homeostatic regulation of neural activity as a therapeutic tool.

BACKGROUND:Huntington's disease (HD) is a progressive neurodegenerative disorder associated with aging, caused by an expanded polyglutamine (polyQ) repeat within the Huntingtin (HTT) protein. In HD, degeneration of the striatum and atrophy of the cortex are observed while cerebellum is less affected.OBJECTIVE:To test the hypothesis that HTT protein levels decline with age, which together with HTT mutation could influence disease progression.METHODS:Using whole brain cell lysates, a unique method of SDS-PAGE and western analysis was used to quantitate HTT protein, which resolves as a monomer and as a high molecular weight species that is modulated by the presence of transglutaminase 2. HTT levels were measured in striatum, cortex and cerebellum in congenic homozygous Q140 and HdhQ150 knock-in mice and WT littermate controls.RESULTS:Mutant HTT in both homozygous knock-in HD mouse models and WT HTT in control striatal and cortical tissues significantly declined in a progressive manner over time. Levels of mutant HTT in HD cerebellum remained high during aging.CONCLUSIONS:A general decline in mutant HTT levels in striatum and cortex is observed that may contribute to disease progression in homozygous knock-in HD mouse models through reduction of HTT function. In cerebellum, sustained levels of mutant HTT with aging may be protective to this tissue which is less overtly affected in HD.

Background: The polyglutamine (polyQ) stretch of the Huntingtin protein (HTT) in mammals is flanked by a highly conserved 17 amino acid N-terminal domain (N17), and a proline-rich region (PRR). The PRR is a binding site for many HTT-interacting proteins, and the N17 domain regulates several normal HTT functions, including HTT’s ability to associate with membranes and organelles. Objective: This study investigates the consequence of deleting mouse Huntingtin’s (Htt’s) N17 domain or a combination of its polyQ stretch and PRR (QP) on normal Htt function in mice. Methods: Knock-in mice expressing versions of Htt lacking either the N17 domain (HttΔN17) or both the polyQ and PRR domains (HttΔQP) were generated, and their behavior, autophagy function, and neuropathology were evaluated. Results: Homozygous and hemizygous HttΔQP/ΔQP, HttΔN17/ΔN17, HttΔQP/–, and HttΔN17/– mice were generated at the expected Mendelian frequency. HttΔQP/ΔQP mutants exhibit improvements in motor coordination compared to controls (Htt+/+). In contrast, HttΔN17/ΔN17 mutants do not exhibit any changes in motor coordination, but they do display variable changes in spatial learning that are dependent on their age at testing. Neither mutant exhibited any changes in basal autophagy in comparison to controls, but thalamostriatal synapses in the dorsal striatum of 24-month-old HttΔN17/ΔN17 mice were decreased compared to controls. Conclusions: These findings support the hypothesis that Htt’s N17 and QP domains are dispensable for its critical functions during early embryonic development, but are likely more important for Htt functions in CNS development or maintenance.

The objective of this study was to characterize the cutaneous antifungal bacterial flora of a plethodontid salamander, Plethodon cinereus, and to relate our results to aspects of amphibian ecology. Scanning electron micrographs of salamander skin indicated a wide distribution of bacteria across the skin. Polymerase Chain Reaction/Denaturant Gradient Gel Electrophoresis (PCR/DGGE) analyses of 16S rRNA gene fragments revealed that a transient community of bacteria was rinsed off and that a resident epibiotic community remained on the skin. Resident bacteria were isolated from the skin of P. cinereus on a low nutrient medium and challenged against a pathogenic ascomycete fungus collected from dead salamander eggs. The strong antifungal bacteria, identified by sequencing of the 16S rRNA gene (similar to 1400 bp), were related to the genera Lysobacter, Pseudomonas, Chryseobacterium, and Bacillus. Patterns of the cutaneous bacterial flora varied among individual salamanders, as revealed by DGGE, although analysis of DGGE bands from salamander skin revealed the presence of at least one species of uncultured beta-proteobacteria on virtually every salamander. These bacteria were closely related to an isolated antifungal Janthinobacterium lividum strain. Antifungal skin bacteria may form mutualistic ecological relationships with amphibian species and help protect them from pathogenic fungi.

Huntington's disease (HD) is a neurodegenerative disorder that effects up to 1 in every 10,000 people and it is caused by an expansion of the polyglutamine (polyQ) stretch within the Huntingtin (HTT) protein [1].The HTT polyQ in mammals is flanked by a highly conserved 17 amino acid N-terminal domain (N17), and a proline-rich region (PRR) [2][3][4].The PRR is a binding site for many , and the N17 domain regulates several normal HTT functions, including HTT's ability to associate with membranes and organelles [8,9].This study investigates the consequence of deleting mouse Huntingtin's (Htt's) N17 domain (ΔN17), deleting a combination of its polyQ stretch and PRR (ΔQP), or deleting all three N-terminal domains (ΔNQP) on normal murine Htt function and in the context of an HD mouse model (Q140).Htt ΔN17 , Htt ΔQP , and Htt ΔNQP knock-in mice were generated, and their behavior, autophagy function, and neuropathology were evaluated.Homozygous and hemizygous Htt ΔN17/ΔN17 , Htt ΔQP/ΔQP , Htt ΔNQP/ΔNQP , Htt ΔN17/-, and Htt ΔQP/- mice were generated at the expected Mendelian frequency.Htt ΔQP/ΔQP mutants exhibit improvements in motor coordination compared to controls (Htt +/+ ) while in contrast, Htt ΔN17/ΔN17 mutants do not exhibit any changes in motor coordination, but they do display variable changes in spatial learning and memory.Neither mutant exhibited any changes in basal autophagy in comparison to controls, but thalamostriatal synapses in the dorsal striatum of 24-month-old Htt ΔN17/ΔN17 mice were decreased compared to controls.These findings support the hypothesis that Htt's N17, polyQ, and PRR domains are dispensable for its critical functions during early embryonic development, but are likely more important for Htt functions in CNS development or maintenance.iii In order to investigate the effects of the N-terminal domain deletions in the context of an HD mouse model, Htt ΔN17 and Htt ΔQP mice were also crossed with Q140 mice to generate Htt 140Q/ΔN17 and Htt 140Q/ΔQP mice.Htt 140Q/ΔN17 and Htt 140Q/ΔQP mice demonstrated exacerbated rotarod and activity cage phenotypes, but improvements in spatial learning and memory.Investigation of primary striatal neurons from Htt 140Q/ΔN17 and Htt 140Q/ΔQP mice exposed a deficit in autophagic flux in comparison to controls that was not observed in primary cortical neurons, suggesting that normal Htt's functions may be somewhat altered in different regions of the brain.Additionally, Htt 140Q/ΔN17 primary striatal and cortical neurons had elevated basal p62 immunostaining.These results suggest that expression of the Htt domain deletions in trans with 140Q-Htt can elicit autophagy phenotypes in cortical and striatal neurons.scientific guidance, technical expertise and continual support.