The pathogenic exon 1 HTT protein is produced by incomplete splicing in Huntington’s disease patients

We have previously shown that exon 1 of the huntingtin gene does not always splice to exon 2 resulting in the production of a small polyadenylated mRNA (HTTexon1) that encodes the highly pathogenic exon 1 HTT protein. The level of this read-through product is proportional to CAG repeat length and is present in all knock-in mouse models of Huntington’s disease (HD) with CAG lengths of 50 and above and in the YAC128 and BACHD mouse models, both of which express a copy of the human HTT gene. We have now developed specific protocols for the quantitative analysis of the transcript levels of HTTexon1 in human tissue and applied these to a series of fibroblast lines and post-mortem brain samples from individuals with either adult-onset or juvenile-onset HD. We found that the HTTexon1 mRNA is present in fibroblasts from juvenile HD patients and can also be readily detected in the sensory motor cortex, hippocampus and cerebellum of post-mortem brains from HD individuals, particularly in those with early onset disease. This finding will have important implications for strategies to lower mutant HTT levels in patients and the design of future therapeutics

Alzheimer's disease markers in the aged sheep (Ovis aries)

This study reports the identification and characterization of markers of Alzheimer's disease (AD) in aged sheep (Ovis aries) as a preliminary step toward making a genetically modified large animal model of AD. Importantly, the sequences of key proteins involved in AD pathogenesis are highly conserved between sheep and human. The processing of the amyloid-β (Aβ) protein is conserved between sheep and human, and sheep Aβ1–42/Aβ1–40 ratios in cerebrospinal fluid (CSF) are also very similar to human. In addition, total tau and neurofilament light levels in CSF are comparable with those found in human. The presence of neurofibrillary tangles in aged sheep brain has previously been established; here, we report for the first time that plaques, the other pathologic hallmark of AD, are also present in the aged sheep brain. In summary, the biological machinery to generate the key neuropathologic features of AD is conserved between the human and sheep, making the sheep a good candidate for future genetic manipulation to accelerate the condition for use in pathophysiological discovery and therapeutic testing

Chemical neuroanatomy of the substantia nigra in the ovine brain

The substantia nigra is an integral component of the basal ganglia circuitry for limbic and motor functions. Dysfunction and degeneration of the basal ganglia are fundamental aspects of neurodegenerative diseases such as Parkinson’s disease and Huntington’s disease. With the increasing use of sheep to model neurological diseases, it is crucial to understand the anatomy and neurochemistry of these key basal ganglia nuclei in the normal sheep brain and how they compare to the human brain. Therefore, studies of the gross anatomy, cellular morphology, and neurochemical expression patterns within the sheep substantia nigra were performed. We show that the sheep substantia nigra reflects all important aspects of the anatomy and neurochemistry of the human substantia nigra, with only minor inter-species differences evident. Many neurochemicals that are central to the functioning of the SN, and wider basal ganglia circuitry, are present throughout the sheep SN. In a wider context, the results of this study provide evidence that the sheep substantia nigra accurately reflects the anatomy of the human substantia nigra, which validates the use of sheep models of basal ganglia neurological disorders

The histamine H4 receptor is functionally expressed on neurons in the mammalian CNS

BACKGROUND AND PURPOSE: The histamine H(4) receptor is the most recently identified of the G protein-coupled histamine receptor family and binds several neuroactive drugs, including amitriptyline and clozapine. So far, H(4) receptors have been found only on haematopoietic cells, highlighting its importance in inflammatory conditions. Here we investigated the possibility that H(4) receptors may be expressed in both the human and mouse CNS. METHODS: Immunological and pharmacological studies were performed using a novel anti-H(4) receptor antibody in both human and mouse brains, and electrophysiological techniques in the mouse brain respectively. Pharmacological tools, selective for the H(4) receptor and patch clamp electrophysiology, were utilized to confirm functional properties of the H(4) receptor in layer IV of the mouse somatosensory cortex. RESULTS: Histamine H(4) receptors were prominently expressed in distinct deep laminae, particularly layer VI, in the human cortex, and mouse thalamus, hippocampal CA4 stratum lucidum and layer IV of the cerebral cortex. In layer IV of the mouse somatosensory cortex, the H(4) receptor agonist 4-methyl histamine (20 µmol·L(−1)) directly hyperpolarized neurons, an effect that was blocked by the selective H(4) receptor antagonist JNJ 10191584, and promoted outwardly rectifying currents in these cells. Monosynaptic thalamocortical CNQX-sensitive excitatory postsynaptic potentials were not altered by 4-methyl histamine (20 µmol·L(−1)) suggesting that H(4) receptors did not act as hetero-receptors on thalamocortical glutamatergic terminals. CONCLUSIONS AND IMPLICATIONS: This is the first demonstration that histamine H(4) receptors are functionally expressed on neurons, which has major implications for the therapeutic potential of these receptors in neurology and psychiatry

PU.1 regulates Alzheimer's disease-associated genes in primary human microglia

Background Microglia play critical roles in the brain during homeostasis and pathological conditions. Understanding the molecular events underpinning microglial functions and activation states will further enable us to target these cells for the treatment of neurological disorders. The transcription factor PU.1 is critical in the development of myeloid cells and a major regulator of microglial gene expression. In the brain, PU.1 is specifically expressed in microglia and recent evidence from genome-wide association studies suggests that reductions in PU.1 contribute to a delayed onset of Alzheimer’s disease (AD), possibly through limiting neuroinflammatory responses. Methods To investigate how PU.1 contributes to immune activation in human microglia, microarray analysis was performed on primary human mixed glial cultures subjected to siRNA-mediated knockdown of PU.1. Microarray hits were confirmed by qRT-PCR and immunocytochemistry in both mixed glial cultures and isolated microglia following PU.1 knockdown. To identify attenuators of PU.1 expression in microglia, high throughput drug screening was undertaken using a compound library containing FDA-approved drugs. NanoString and immunohistochemistry was utilised to investigate the expression of PU.1 itself and PU.1-regulated mediators in primary human brain tissue derived from neurologically normal and clinically and pathologically confirmed cases of AD. Results Bioinformatic analysis of gene expression upon PU.1 silencing in mixed glial cultures revealed a network of modified AD-associated microglial genes involved in the innate and adaptive immune systems, particularly those involved in antigen presentation and phagocytosis. These gene changes were confirmed using isolated microglial cultures. Utilising high throughput screening of FDA-approved compounds in mixed glial cultures we identified the histone deacetylase inhibitor vorinostat as an effective attenuator of PU.1 expression in human microglia. Further characterisation of vorinostat in isolated microglial cultures revealed gene and protein changes partially recapitulating those seen following siRNA-mediated PU.1 knockdown. Lastly, we demonstrate that several of these PU.1-regulated genes are expressed by microglia in the human AD brain in situ. Conclusions Collectively, these results suggest that attenuating PU.1 may be a valid therapeutic approach to limit microglial-mediated inflammatory responses in AD and demonstrate utility of vorinostat for this purpose

Huntington's disease mice and human brain tissue exhibit increased G3BP1 granules and TDP43 mislocalization

Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington's disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of HD patients and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex of HD patients. Of these targets, SG components were enriched, including the SG nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of HD patient brains. Intriguingly, we also observed that the SG-associated TAR DNA-Binding Protein-43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1-granule positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD