Search

Abstract Gingipains are protease virulence factors produced by Porphyromonas gingivalis, a Gram-negative bacterium best known for its role in chronic periodontitis. Gingipains were recently identified in the middle temporal gyrus of postmortem Alzheimer’s disease (AD) brains, where gingipain load correlated with AD diagnosis and tau and ubiquitin pathology. Since AD and Parkinson’s disease (PD) share some overlapping pathologic features, including nigral pathology and Lewy bodies, the current study explored whether gingipains are present in the substantia nigra pars compacta of PD brains. In immunohistochemical techniques and multi-channel fluorescence studies, gingipain antigens were abundant in dopaminergic neurons in the substantia nigra of both PD and neurologically normal control brains. 3-dimensional reconstructions of Lewy body containing neurons revealed that gingipains associated with the periphery of alpha-synuclein aggregates but were occasionally observed inside aggregates. In vitro proteomic analysis demonstrated that recombinant alpha-synuclein is cleaved by lysine-gingipain, generating multiple alpha-synuclein fragments including the non-amyloid component fragments. Immunogold electron microscopy with co-labeling of gingipains and alpha-synuclein confirmed the occasional colocalization of gingipains with phosphorylated (pSER129) alpha-synuclein. In dopaminergic neurons, gingipains localized to the perinuclear cytoplasm, neuromelanin, mitochondria, and nucleus. These data suggest that gingipains localize in dopaminergic neurons in the substantia nigra and interact with alpha-synuclein.

Abstract Understanding the mechanisms that drive TDP-43 pathology is integral to combating amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and other neurodegenerative diseases. Here we generated a longitudinal quantitative proteomic map of the cortex from the cytoplasmic TDP-43 rNLS8 mouse model of ALS and FTLD, and developed a complementary open-access webtool, TDP-map ( https://shiny.rcc.uq.edu.au/TDP-map/ ). We identified distinct protein subsets enriched for diverse biological pathways with temporal alterations in protein abundance, including increases in protein folding factors prior to disease onset. This included increased levels of DnaJ homolog subfamily B member 5, DNAJB5, which also co-localized with TDP-43 pathology in diseased human motor cortex. DNAJB5 over-expression decreased TDP-43 aggregation in cell and cortical neuron cultures, and knockout of Dnajb5 exacerbated motor impairments caused by AAV-mediated cytoplasmic TDP-43 expression in mice. Together, these findings reveal molecular mechanisms at distinct stages of ALS and FTLD progression and suggest that protein folding factors could be protective in neurodegenerative diseases.

Abstract Microglia, the innate immune cells of the brain, are activated by damage or disease. In mouse models of amyotrophic lateral sclerosis (ALS), microglia shift from neurotrophic to neurotoxic states with disease progression. It remains unclear how human microglia change relative to the TAR DNA-binding protein 43 (TDP-43) aggregation that occurs in 97% of ALS cases. Here we examine spatial relationships between microglial activation and TDP-43 pathology in brain tissue from people with ALS and from a TDP-43-driven ALS mouse model. Post-mortem human brain tissue from the Neurological Foundation Human Brain Bank was obtained from 10 control and 10 ALS cases in parallel with brain tissue from a bigenic NEFH-tTA/tetO-hTDP-43∆NLS (rNLS) mouse model of ALS at disease onset, early disease, and late disease stages. The spatiotemporal relationship between microglial activation and ALS pathology was determined by investigating microglial functional marker expression in brain regions with low and high TDP-43 burden at end-stage human disease: hippocampus and motor cortex, respectively. Sections were immunohistochemically labelled with a two-round multiplexed antibody panel against; microglial functional markers (L-ferritin, HLA-DR, CD74, CD68, and Iba1), a neuronal marker, an astrocyte marker, and pathological phosphorylated TDP-43 (pTDP-43). Single-cell levels of microglial functional markers were quantified using custom analysis pipelines and mapped to anatomical regions and ALS pathology. We identified a significant increase in microglial Iba1 and CD68 expression in the human ALS motor cortex, with microglial CD68 being significantly correlated with pTDP-43 pathology load. We also identified two subpopulations of microglia enriched in the ALS motor cortex that were defined by high L-ferritin expression. A similar pattern of microglial changes was observed in the rNLS mouse, with an increase first in CD68 and then in L-ferritin expression, with both occurring only after pTDP-43 inclusions were detectable. Our data strongly suggest that microglia are phagocytic at early-stage ALS but transition to a dysfunctional state at end-stage disease, and that these functional states are driven by pTDP-43 aggregation. Overall, these findings enhance our understanding of microglial phenotypes and function in ALS.

Abstract Parkinson’s disease (PD) is characterised by the progressive loss of midbrain dopaminergic neurons and the presence of aggregated α-synuclein (α-syn). Pericytes and microglia, two non-neuronal cells contain α-syn in the human brain, however, their role in disease processes is poorly understood. Pericytes, found surrounding the capillaries in the brain are important for maintaining the blood–brain barrier, controlling blood flow and mediating inflammation. In this study, primary human brain pericytes and microglia were exposed to two different α-synuclein aggregates. Inflammatory responses were assessed using immunocytochemistry, cytometric bead arrays and proteome profiler cytokine array kits. Fixed flow cytometry was used to investigate the uptake and subsequent degradation of α-syn in pericytes. We found that the two α-syn aggregates are devoid of inflammatory and cytotoxic actions on human brain derived pericytes and microglia. Although α-syn did not induce an inflammatory response, pericytes efficiently take up and degrade α-syn through the lysosomal pathway but not the ubiquitin–proteasome system. Furthermore, when pericytes were exposed the ubiquitin proteasome inhibitor—MG132 and α-syn aggregates, there was profound cytotoxicity through the production of reactive oxygen species resulting in apoptosis. These results suggest that the observed accumulation of α-syn in pericytes in human PD brains likely plays a role in PD pathogenesis, perhaps by causing cerebrovascular instability, under conditions of cellular stress.

Smyth et al. use tissue microarrays from Alzheimer’s disease (AD) patient brains to show that PDGF-BB:PDGFRβ signalling components are reduced in AD. They then use primary human brain pericytes to elucidate a pathway by which PDGF-BB:PDGFRβ signalling in brain pericytes is disrupted in AD, thus impairing the blood brain barrier.

Abstract Introduction Neutrophil accumulation is a well-established feature of Alzheimer’s disease (AD) and has been linked to cognitive impairment by modulating disease-relevant neuroinflammatory and vascular pathways. Neutrophils express high levels of the oxidant-generating enzyme myeloperoxidase (MPO), however there has been controversy regarding the cellular source and localisation of MPO in the AD brain. Materials and methods We used immunostaining and immunoassays to quantify the accumulation of neutrophils in human AD tissue microarrays and in the brains of APP/PS1 mice. We also used multiplexed immunolabelling to define the presence of NETs in AD. Results There was an increase in neutrophils in AD brains as well as in the murine APP/PS1 model of AD. Indeed, MPO expression was almost exclusively confined to S100A8-positive neutrophils in both human AD and murine APP/PS1 brains. The vascular localisation of neutrophils in both human AD and mouse models of AD was striking and driven by enhanced neutrophil adhesion to small vessels. We also observed rare infiltrating neutrophils and deposits of MPO around plaques. Citrullinated histone H3, a marker of neutrophil extracellular traps (NETs), was also detected in human AD cases at these sites, indicating the presence of extracellular MPO in the vasculature. Finally, there was a reduction in the endothelial glycocalyx in AD that may be responsible for non-productive neutrophil adhesion to the vasculature. Conclusion Our report indicates that vascular changes may drive neutrophil adhesion and NETosis, and that neutrophil-derived MPO may lead to vascular oxidative stress and be a relevant therapeutic target in AD.

Murray et al. present a multiplexed fluorescence-based immunohistochemistry approach and use it to screen up to 100 protein markers in the human olfactory bulb from neurologically healthy, Alzheimer’s, and Parkinson’s disease patients. In doing so, they identify differentially expressed biomarkers in Alzheimer’s, and Parkinson’s disease.

The study of microglia isolated from adult human brain tissue provides unique insight into the physiology of these brain immune cells and their role in adult human brain disorders. Reports of microglia in post-mortem adult human brain tissue show regional differences in microglial populations, however, these differences have not been fully explored in living microglia. In this study biopsy tissue was obtained from epileptic patients undergoing surgery and consisted of both cortical areas and neurogenic ventricular and hippocampal (Hp) areas. Microglia were concurrently isolated from both regions and compared by immunochemistry. Our initial observation was that a greater number of microglia resulted from isolation and culture of ventricular/Hp tissue than cortical tissue. This was found to be due to a greater proliferative capacity of microglia from ventricular/Hp regions compared to the cortex. Additionally, ventricular/Hp microglia had a greater proliferative response to the microglial mitogen Macrophage Colony-Stimulating Factor (M-CSF). This enhanced response was found to be associated with higher M-CSF receptor expression and higher expression of proteins involved in M-CSF signalling DAP12 and C/EBPβ. Microglia from the ventricular/Hp region also displayed higher expression of the receptor for Insulin-like Growth Factor-1, a molecule with some functional similarity to M-CSF. Compared to microglia isolated from the cortex, ventricular/Hp microglia showed increased HLA-DP, DQ, DR antigen presentation protein expression and a rounded morphology. These findings show that microglia from adult human brain neurogenic regions are more proliferative than cortical microglia and have a distinct protein expression profile. The data present a case for differential microglial phenotype and function in different regions of the adult human brain and suggest that microglia in adult neurogenic regions are “primed” to an activated state by their unique tissue environment.

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving progressive degeneration of upper and lower motor neurons. The pattern of lower motor neuron loss along the spinal cord follows the pattern of deposition of phosphorylated TDP-43 aggregates. The blood-spinal cord barrier (BSCB) restricts entry into the spinal cord parenchyma of blood components that can promote motor neuron degeneration, but in ALS there is evidence for barrier breakdown. Here we sought to quantify BSCB breakdown along the spinal cord axis, to determine whether BSCB breakdown displays the same patterning as motor neuron loss and TDP-43 proteinopathy. Cerebrospinal fluid hemoglobin was measured in living ALS patients (n = 87 control, n = 236 ALS) as a potential biomarker of BSCB and blood–brain barrier leakage. Cervical, thoracic, and lumbar post-mortem spinal cord tissue (n = 5 control, n = 13 ALS) were then immunolabelled and semi-automated imaging and analysis performed to quantify hemoglobin leakage, lower motor neuron loss, and phosphorylated TDP-43 inclusion load. Hemoglobin leakage was observed along the whole ALS spinal cord axis and was most severe in the dorsal gray and white matter in the thoracic spinal cord. In contrast, motor neuron loss and TDP-43 proteinopathy were seen at all three levels of the ALS spinal cord, with most abundant TDP-43 deposition in the anterior gray matter of the cervical and lumbar cord. Our data show that leakage of the BSCB occurs during life, but at end-stage disease the regions with most severe BSCB damage are not those where TDP-43 accumulation is most abundant. This suggests BSCB leakage and TDP-43 pathology are independent pathologies in ALS.

Glutamate is the main excitatory neurotransmitter in the human central nervous system, responsible for a wide variety of normal physiological processes. Glutamatergic metabolism and its sequestration are tightly regulated in the normal human brain, and it has been demonstrated that dysregulation of the glutamatergic system can have wide-ranging effects both in acute brain injury and neurodegenerative diseases. The excitatory amino acid transporter 2 (EAAT2) is the dominant glutamatergic transporter in the human brain, responsible for efficient removal of glutamate from the synaptic cleft for recycling within glial cells. As such, it has a key role in maintaining excitatory-inhibitory homeostasis. Animal studies have demonstrated dysregulation or alterations of EAAT2 expression can have implications in neurodegenerative disorders. Despite extensive research into glutamatergic alterations in AD mouse models, there is a lack of studies examining the expression of EAAT2 within the AD human brain. In this systematic review, 29 articles were identified that either analyzed EAAT2 expression in the AD human brain or used a human-derived cell culture. Studies were inconclusive as to whether EAAT2 was upregulated or downregulated in AD. However, changes in localization and correlation between EAAT2 expression and symptomatology was noted. These findings implicate EAAT2 alterations as a key process in AD progression and highlight the need for further research into the characterization of EAAT2 processes in normal physiology and disease in human tissue and to identify compounds that can act as EAAT2 neuromodulators.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease associated with a history of repetitive head impacts (RHI). CTE was described in boxers as early as the 1920s and by the 1950s it was widely accepted that hits to the head caused some boxers to become “punch drunk.” However, the recent discovery of CTE in American and Australian-rules football, soccer, rugby, ice hockey, and other sports has resulted in renewed debate on whether the relationship between RHI and CTE is causal. Identifying the strength of the evidential relationship between CTE and RHI has implications for public health and medico-legal issues. From a public health perspective, environmentally caused diseases can be mitigated or prevented. Medico-legally, millions of children are exposed to RHI through sports participation; this demographic is too young to legally consent to any potential long-term risks associated with this exposure. To better understand the strength of evidence underlying the possible causal relationship between RHI and CTE, we examined the medical literature through the Bradford Hill criteria for causation. The Bradford Hill criteria, first proposed in 1965 by Sir Austin Bradford Hill, provide a framework to determine if one can justifiably move from an observed association to a verdict of causation. The Bradford Hill criteria include nine viewpoints by which to evaluate human epidemiologic evidence to determine if causation can be deduced: strength, consistency, specificity, temporality, biological gradient, plausibility, coherence, experiment, and analogy. We explored the question of causation by evaluating studies on CTE as it relates to RHI exposure. Through this lens, we found convincing evidence of a causal relationship between RHI and CTE, as well as an absence of evidence-based alternative explanations. By organizing the CTE literature through this framework, we hope to advance the global conversation on CTE mitigation efforts.

Omeprazole is extensively used to manage gastroesophageal reflux disease (GERD). It is primarily metabolized by CYP2C19. The CYP2C19*17 (rs12248560) allele and the recently described CYP2C:TG haplotype (rs11188059 and rs2860840) are associated with increased enzymatic activity, and may reduce omeprazole exposure. This observational study aimed to investigate the association between these genetic variants and omeprazole treatment failure in GERD. We recruited predominantly New Zealand European GERD patients who either did not respond to omeprazole or experienced breakthrough heartburn symptoms despite at least 8 weeks of omeprazole (≥40 mg/day). The GerdQ score was used to gauge symptomatic severity. A total of 55 cases were recruited with a median age (range) of 56 years (19–82) and GerdQ score of 11 (5–17). Of these, 19 (34.5%) were CYP2C19*17 heterozygotes and two (3.6%) were CYP2C19*17 homozygotes. A total of 30 (27.3%) CYP2C:TG haplotypes was identified in our cohort, with seven (12.7%) CYP2C:TG homozygotes, and 16 (29%) CYP2C:TG heterozygotes. No significant differences were observed for overall CYP2C19*17 alleles, CYP2C19*17/*17, overall CYP2C:TG haplotypes, and CYP2C:TG heterozygotes (p > 0.05 for all comparisons). Gastroscopy and 24-h esophageal pH/impedance tests demonstrated objective evidence of GERD in a subgroup of 39 (71%) cases, in which the CYP2C:TG/TG was significantly enriched (p = 0.03) when compared with the haplotype frequencies in a predominantly (91%) New Zealand European reference population, but not the CYP2C19*17/*17 (p > 0.99), when compared with the allele frequencies for the non-Finnish European subset of gnomAD. We conclude that omeprazole treatment failure in GERD is associated with CYP2C:TG/TG, but not CYP2C19*17.

Huntington’s disease (HD) is an autosomal neurodegenerative disease that is characterized by an excessive number of CAG trinucleotide repeats within the huntingtin gene ( HTT). HD patients can present with a variety of symptoms including chorea, behavioural and psychiatric abnormalities and cognitive decline. Each patient has a unique combination of symptoms, and although these can be managed using a range of medications and non-drug treatments there is currently no cure for the disease. Current therapies prescribed for HD can be categorized by the symptom they treat. These categories include chorea medication, antipsychotic medication, antidepressants, mood stabilizing medication as well as non-drug therapies. Fortunately, there are also many new HD therapeutics currently undergoing clinical trials that target the disease at its origin; lowering the levels of mutant huntingtin protein (mHTT). Currently, much attention is being directed to antisense oligonucleotide (ASO) therapies, which bind to pre-RNA or mRNA and can alter protein expression via RNA degradation, blocking translation or splice modulation. Other potential therapies in clinical development include RNA interference (RNAi) therapies, RNA targeting small molecule therapies, stem cell therapies, antibody therapies, non-RNA targeting small molecule therapies and neuroinflammation targeted therapies. Potential therapies in pre-clinical development include Zinc-Finger Protein (ZFP) therapies, transcription activator-like effector nuclease (TALEN) therapies and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) therapies. This comprehensive review aims to discuss the efficacy of current HD treatments and explore the clinical trial progress of emerging potential HD therapeutics.

Abstract In Alzheimer’s disease (AD), microglia are affected by disease processes, but may also drive pathogenesis. AD pathology-associated microglial populations have been identified with single-cell RNA-Seq, but have not been validated in human brain tissue with anatomical context. Here, we quantified myeloid cell markers to identify changes in AD pathology-associated microglial populations. We performed fluorescent immunohistochemistry on normal (n = 8) and AD (n = 8) middle temporal gyri, co-labelling the pan-myeloid cell marker, Iba1, with one of 11 markers of interest (MOIs): CD45, HLA-DR, CD14, CD74, CD33, CD206, CD32, CD163, P2RY12, TMEM119, L-Ferritin. Novel image analyses quantified the single-cell abundance of Iba1 and each MOI. Each cell was gated into one Iba1-MOI population (Iba1low MOIhigh, Iba1high MOIhigh, or Iba1high MOIlow) and the abundance of each population was compared between AD and control. Triple-labelling of L-Ferritin and Iba1 with a subset of MOIs was performed to investigate L-Ferritin-MOI co-expression on Iba1low cells. Iba1low MOIhigh myeloid cell populations delineated by MOIs CD45, HLA-DR, CD14, CD74, CD33, CD32, and L-Ferritin were increased in AD. Further investigation of the Iba1low MOIhigh populations revealed that their abundances correlated with tau, but not amyloid beta, load in AD. The Iba1low microglial population highly expressed L-Ferritin, reflecting microglial dysfunction. The L-Ferritinhigh CD74high HLA-DRhigh phenotype of the Iba1low population mirrors that of a human AD pathology-associated microglial subpopulation previously identified using single-cell RNA-Seq. Our high-throughput immunohistochemical data with anatomical context support the microglial dysfunction hypothesis of AD.

Abstract Olfactory dysfunction is an early and prevalent symptom of Alzheimer’s disease (AD) and the olfactory bulb is a nexus of beta-amyloid plaque and tau neurofibrillary tangle (NFT) pathology during early AD progression. To mitigate the accumulation of misfolded proteins, an endoplasmic reticulum stress response called the unfolded protein response (UPR) occurs in the AD hippocampus. However, chronic UPR activation can lead to apoptosis and the upregulation of beta-amyloid and tau production. Therefore, UPR activation in the olfactory system could be one of the first changes in AD. In this study, we investigated whether two proteins that signal UPR activation are expressed in the olfactory system of AD cases with low or high amounts of aggregate pathology. We used immunohistochemistry to label two markers of UPR activation (p-PERK and p-eIF2α) concomitantly with neuronal markers (NeuN and PGP9.5) and pathology markers (beta-amyloid and tau) in the olfactory bulb, piriform cortex, entorhinal cortex and the CA1 region of the hippocampus in AD and normal cases. We show that UPR activation, as indicated by p-PERK and p-eIF2α expression, is significantly increased throughout the olfactory system in AD cases with low (Braak stage III-IV) and high-level (Braak stage V-VI) pathology. We further show that UPR activation occurs in the mitral cells and in the anterior olfactory nucleus of the olfactory bulb where tau and amyloid pathology is abundant. However, UPR activation is not present in neurons when they contain NFTs and only rarely occurs in neurons containing diffuse tau aggregates. We conclude that UPR activation is prevalent in all regions of the olfactory system and support previous findings suggesting that UPR activation likely precedes NFT formation. Our data indicate that chronic UPR activation in the olfactory system might contribute to the olfactory dysfunction that occurs early in the pathogenesis of AD.

Neurological diseases including Alzheimer’s, Huntington’s disease, Parkinson’s disease, Down syndrome and epilepsy, and neuropsychiatric disorders such as schizophrenia, are conditions that affect not only individuals but societies on a global scale. Current therapies offer a means for small symptomatic relief, but recently there has been increasing demand for therapeutic alternatives. The γ-aminobutyric acid (GABA)ergic signaling system has been investigated for developing new therapies as it has been noted that any dysfunction or changes to this system can contribute to disease progression. Expression of the K-Cl-2 (KCC2) and N-K-C1-1 (NKCC1) cation-chloride cotransporters (CCCs) has recently been linked to the disruption of GABAergic activity by affecting the polarity of GABAA receptor signaling. KCC2 and NKCC1 play a part in multiple neurological and neuropsychiatric disorders, making them a target of interest for potential therapies. This review explores current research suggesting the pathophysiological role and therapeutic importance of KCC2 and NKCC1 in neuropsychiatric and neurological disorders.

Gene expression studies of human post-mortem brain tissue are useful for understanding the pathogenesis of neurodegenerative disease. These studies rely on the assumption that RNA quality is consistent between disease and neurologically normal cases; however, previous studies have suggested that RNA quality may be affected by neurodegenerative disease. Here, we compared RNA quality in human post-mortem brain tissue between neurologically normal cases (n = 14) and neurodegenerative disease cases (Alzheimer’s disease n = 10; Parkinson’s disease n = 11; and Huntington’s disease n = 9) in regions affected by pathology and regions that are relatively devoid of pathology. We identified a statistically significant decrease in RNA integrity number (RIN) in Alzheimer’s disease tissue relative to neurologically normal tissue (mixed effects model, p = 0.04). There were no statistically significant differences between neurologically normal cases and Parkinson’s disease or Huntington’s disease cases. Next, we investigated whether total RNA quality affected mRNA quantification, by correlating RIN with qPCR threshold cycle (CT). CT values for all six genes investigated were strongly correlated with RIN (p < 0.05, Pearson correlation); this effect was only partially mitigated by normalization to RPL30. Our results indicate that RNA quality is decreased in Alzheimer’s disease tissue. We recommend that RIN should be considered when this tissue is used in gene expression analyses.

Alzheimer’s disease (AD) is a neuropathological disorder characterized by the presence and accumulation of amyloid-beta plaques and neurofibrillary tangles. Glutamate dysregulation and the concept of glutamatergic excitotoxicity have been frequently described in the pathogenesis of a variety of neurodegenerative disorders and are postulated to play a major role in the progression of AD. In particular, alterations in homeostatic mechanisms, such as glutamate uptake, have been implicated in AD. An association with excitatory amino acid transporter 2 (EAAT2), the main glutamate uptake transporter, dysfunction has also been described. Several animal and few human studies examined EAAT2 expression in multiple brain regions in AD but studies of the hippocampus, the most severely affected brain region, are scarce. Therefore, this study aims to assess alterations in the expression of EAAT2 qualitatively and quantitatively through DAB immunohistochemistry (IHC) and immunofluorescence within the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus (STG) regions, between human AD and control cases. Although no significant EAAT2 density changes were observed between control and AD cases, there appeared to be increased transporter expression most likely localized to fine astrocytic branches in the neuropil as seen on both DAB IHC and immunofluorescence. Therefore, individual astrocytes are not outlined by EAAT2 staining and are not easily recognizable in the CA1–3 and dentate gyrus regions of AD cases, but the altered expression patterns observed between AD and control hippocampal cases could indicate alterations in glutamate recycling and potentially disturbed glutamatergic homeostasis. In conclusion, no significant EAAT2 density changes were found between control and AD cases, but the observed spatial differences in transporter expression and their functional significance will have to be further explored.

Alzheimer’s disease (AD) is the leading cause of dementia, predicted to be the most significant health burden of the 21st century, with an estimated 131.5 million dementia patients by the year 2050. This review aims to provide an overview of the effect of caffeine on AD and cognition by summarizing relevant research conducted on this topic. We searched the Web of Science core collection and PubMed for studies related to the effect of caffeine on AD and cognition using title search terms: caffeine; coffee; Alzheimer’s; cognition. There is suggestive evidence from clinical studies that caffeine is neuroprotective against dementia and possibly AD (20 out of 30 studies support this), but further studies, such as the “ideal” study proposed in this review, are required to prove this link. Clinical studies also indicate that caffeine is a cognitive normalizer and not a cognitive enhancer. Furthermore, clinical studies suggest the neuroprotective effect of caffeine might be confounded by gender. There is robust evidence based on in vivo and in vitro studies that caffeine has neuroprotective properties in AD animal models (21 out of 22 studies support this), but further studies are needed to identify the mechanistic pathways mediating these effects.

Alzheimer’s disease (AD) is a neurodegenerative disorder with an increasing need for developing disease-modifying treatments as current therapies only provide marginal symptomatic relief. Recent evidence suggests the γ-aminobutyric acid (GABA) neurotransmitter system undergoes remodeling in AD, disrupting the excitatory/inhibitory (E/I) balance in the brain. Altered expression levels of K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1), which are cation–chloride cotransporters (CCCs), have been implicated in disrupting GABAergic activity by regulating GABAA receptor signaling polarity in several neurological disorders, but these have not yet been explored in AD. NKCC1 and KCC2 regulate intracellular chloride [Cl−]i by accumulating and extruding Cl−, respectively. Increased NKCC1 expression in mature neurons has been reported in these disease conditions, and bumetanide, an NKCC1 inhibitor, is suggested to show potential therapeutic benefits. This study used primary mouse hippocampal neurons to explore if KCC2 and NKCC1 expression levels are altered following beta-amyloid (Aβ1-42) treatment and the potential neuroprotective effects of bumetanide. KCC2 and NKCC1 expression levels were also examined in 18-months-old male C57BL/6 mice following bilateral hippocampal Aβ1-42 stereotaxic injection. No change in KCC2 and NKCC1 expression levels were observed in mouse hippocampal neurons treated with 1 nM Aβ1-42, but NKCC1 expression increased 30-days post-Aβ1-42-injection in the CA1 region of the mouse hippocampus. Primary mouse hippocampal cultures were treated with 1 nM Aβ1-42 alone or with various concentrations of bumetanide (1 µM, 10 µM, 100 µM, 1 mM) to investigate the effect of the drug on cell viability. Aβ1-42 produced 53.1 ± 1.4% cell death after 5 days, and the addition of bumetanide did not reduce this. However, the drug at all concentrations significantly reduced cell viability, suggesting bumetanide is highly neurotoxic. In summary, these results suggest that chronic exposure to Aβ1-42 alters the balance of KCC2 and NKCC1 expression in a region-and layer-specific manner in mouse hippocampal tissue; therefore, this process most likely contributes to altered hippocampal E/I balance in this model. Furthermore, bumetanide induces hippocampal neurotoxicity, thus questioning its suitability for AD therapy. Further investigations are required to examine the effects of Aβ1-42 on KCC2 and NKCC1 expression and whether targeting CCCs might offer a therapeutic approach for AD.

Olfactory deficits are an early and prevalent non-motor symptom of Huntington’s disease (HD). In other neurodegenerative diseases where olfactory deficits occur, such as Alzheimer’s disease and Parkinson’s disease, pathological protein aggregates (tau, β-amyloid, α-synuclein) accumulate in the anterior olfactory nucleus (AON) of the olfactory bulb (OFB). Therefore, in this study we determined whether aggregates are also present in HD OFBs; 13 HD and five normal human OFBs were stained for mutant huntingtin (mHtt), tau, β-amyloid, TDP-43, and α-synuclein. Our results show that mHtt aggregates detected with 1F8 antibody are present within all HD OFBs, and mHtt aggregate load in the OFB does not correlate with Vonsattel grading scores. The majority of the aggregates were located in the AON and in similar abundance in each anatomical segment of the AON. No mHtt aggregates were found in controls; 31% of HD cases also contained tau neurofibrillary tangles within the AON. This work demonstrates HD pathology in the OFB and indicates that disease-specific protein aggregation in the AON is a common feature of neurodegenerative diseases that show olfactory deficits.

Alzheimer’s disease (AD) is the leading type of dementia worldwide. Despite an increasing burden of disease due to a rapidly aging population, there is still a lack of complete understanding of the precise pathological mechanisms which drive its progression. Glutamate is the main excitatory neurotransmitter in the brain and plays an essential role in the normal function and excitability of neuronal networks. While previous studies have shown alterations in the function of the glutamatergic system in AD, the underlying etiology of beta amyloid (Aβ1–42) induced changes has not been explored. Here we have investigated the acute effects of stereotaxic hippocampal Aβ1–42 injection on specific glutamatergic receptors and transporters in the mouse hippocampus, using immunohistochemistry and confocal microscopy 3 days after Aβ1–42 injection in aged male C57BL/6 mice, before the onset of neuronal cell death. We show that acute injection of Aβ1–42 is sufficient to induce cognitive deficits 3 days post-injection. We also report no significant changes in glutamate receptor subunits GluA1, GluA2, VGluT1, and VGluT2 in response to acute injection of Aβ1–42 when compared with the ACSF-vehicle injected mice. However, we observed increased expression in the DG hilus and ventral stratum (str.) granulosum, CA3 str. radiatum and str. oriens, and CA1 str. radiatum of the GluN1 subunit, and increased expression within the CA3 str. radiatum and decreased expression within the DG str. granulosum of the GluN2A subunit in Aβ1–42 injected mice compared to NC, and a similar trend observed when compared to ACSF-injected mice. We also observed alterations in expression patterns of glutamatergic receptor subunits and transporters within specific layers of hippocampal subregions in response to a microinjection stimulus. These findings indicate that the pathological alterations in the glutamatergic system observed in AD are likely to be partially a result of both acute and chronic exposure to Aβ1–42 and implies a much more complex circuit mechanism associated with glutamatergic dysfunction than simply glutamate-mediated excitotoxic neuronal death.

Abstract 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.

Abstract Background Pericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed. Methods To study the effects of neuroinflammation at the BBB, primary brain endothelial cells and pericytes were isolated from human biopsy tissue. Culture purity was examined using qPCR and immunocytochemistry. Electrical cell-substrate impedance sensing (ECIS) was used to determine the barrier properties of endothelial and pericyte cultures. Using immunocytochemistry, cytometric bead array, and ECIS, we compared the responses of endothelia and pericytes to a panel of inflammatory stimuli (IL-1β, TNFα, LPS, IFN-γ, TGF-β1, IL-6, and IL-4). Secretome analysis was performed to identify unique secretions of endothelia and pericytes in response to IL-1β. Results Endothelial cells were pure, moderately proliferative, retained the expression of BBB-related junctional proteins and transporters, and generated robust TEER. Both endothelia and pericytes have the same pattern of transcription factor activation in response to inflammatory stimuli but respond differently at the secretion level. Secretome analysis confirmed that endothelia and pericytes have overlapping but distinct secretome profiles in response to IL-1β. We identified several cell-type specific responses, including G-CSF and GM-CSF (endothelial-specific), and IGFBP2 and IGFBP3 (pericyte-specific). Finally, we demonstrated that direct addition of IL-1β, TNFα, LPS, and IL-4 contributed to the loss of endothelial barrier integrity in vitro. Conclusions Here, we identify important cell-type differences in the inflammatory response of brain pericytes and endothelia and provide, for the first time, a comprehensive profile of the secretions of primary human brain endothelia and pericytes which has implications for understanding how inflammation affects the cerebrovasculature.

Abstract 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.

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease. HD patients present with movement disorders, behavioral and psychiatric symptoms and cognitive decline. This review summarizes the contribution of microglia and astrocytes to HD pathophysiology. Neuroinflammation in the HD brain is characterized by a reactive morphology in these glial cells. Microglia and astrocytes are critical in regulating neuronal activity and maintaining an optimal milieu for neuronal function. Previous studies provide evidence that activated microglia and reactive astrocytes contribute to HD pathology through transcriptional activation of pro-inflammatory genes to perpetuate a chronic inflammatory state. Reactive astrocytes also display functional changes in glutamate and ion homeostasis and energy metabolism. Astrocytic and microglial changes may further contribute to the neuronal death observed with the progression of HD. Importantly, the degree to which these neuroinflammatory changes are detrimental to neurons and contribute to the progression of HD pathology is not well understood. Furthermore, recent observations provide compelling evidence that activated microglia and astrocytes exert a variety of beneficial functions that are essential for limiting tissue damage and preserving neuronal function in the HD brain. Therefore, a better understanding of the neuroinflammatory environment in the brain in HD may lead to the development of targeted and innovative therapeutic opportunities.

Huntington’s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in exon 1 of the HTT gene. HD usually manifests in mid-life with loss of GABAergic projection neurons from the striatum accompanied by progressive atrophy of the putamen followed by other brain regions, but linkages between the genetics and neurodegeneration are not understood. We measured metabolic perturbations in HD-human brain in a case-control study, identifying pervasive lowering of vitamin B5, the obligatory precursor of coenzyme A (CoA) that is essential for normal intermediary metabolism. Cerebral pantothenate deficiency is a newly-identified metabolic defect in human HD that could potentially: (i) impair neuronal CoA biosynthesis; (ii) stimulate polyol-pathway activity; (iii) impair glycolysis and tricarboxylic acid cycle activity; and (iv) modify brain-urea metabolism. Pantothenate deficiency could lead to neurodegeneration/dementia in HD that might be preventable by treatment with vitamin B5.