ABN78 Sigma-AldrichAnti-NeuN Antibody (rabbit)

Hutchinson-Gilford progeria syndrome (HGPS) is a segmental progeroid syndrome with multiple features suggestive of premature accelerated aging. Accumulation of progerin is thought to underlie the pathophysiology of HGPS. However, despite ubiquitous expression of lamin A in all differentiated cells, the HGPS mutation results in organ-specific defects. For example, bone and skin are strongly affected by HGPS, while the brain appears to be unaffected. There are no definite explanations as to the variable sensitivity to progeria disease among different organs. In addition, low levels of progerin have also been found in several tissues from normal individuals, but it is not clear if low levels of progerin contribute to the aging of the brain. In an attempt to clarify the origin of this phenomenon, we have developed an inducible transgenic mouse model with expression of the most common HGPS mutation in brain, skin, bone and heart to investigate how the mutation affects these organs. Ultrastructural analysis of neuronal nuclei after 70 weeks of expression of the LMNA c.1824Cgreater than T mutation showed severe distortion with multiple lobulations and irregular extensions. Despite severe distortions in the nuclei of hippocampal neurons of HGPS animals, there were only negligible changes in gene expression after 63 weeks of transgenic expression. Behavioral analysis and neurogenesis assays, following long-term expression of the HGPS mutation, did not reveal significant pathology. Our results suggest that certain tissues are protected from functional deleterious effects of progerin.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2-12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.

Parkinson's disease is a neurodegenerative disorder characterized by motor and non-motor disturbances. Various pathogenic pathways drive disease progression including oxidative stress, mitochondrial dysfunction, α-synuclein aggregation and impairment of protein degradation systems. Dysfunction of the ubiquitin-proteasome system in the substantia nigra of Parkinson's disease patients is believed to be one of the causes of protein aggregation and cell death associated with this disorder. Lactacystin, a potent inhibitor of the proteasome, was previously delivered to the nigrostriatal pathway of rodents to model nigrostriatal degeneration. Although lactacystin-treated animals develop parkinsonian motor impairment, it is currently unknown whether they also develop non-motor symptoms characteristic of this disorder. In order to further describe the proteasome inhibition model of Parkinson's disease, we characterized the unilateral lactacystin model, performed by stereotaxic injection of the toxin in the substantia nigra of mice. We studied the degree of neurodegeneration and the behavioral phenotype 1 and 3 weeks after lactacystin lesion both in terms of motor impairment, as well as non-motor symptoms. We report that unilateral administration of 3 μg lactacystin to the substantia nigra of mice leads to partial (~40%) dopaminergic cell loss and concurrent striatal dopamine depletion, accompanied by increased expression of Ser129-phosphorylated α-synuclein. Behavioral characterization of the model revealed parkinsonian motor impairment, as well as signs of non-motor disturbances resembling early stage Parkinson's disease including sensitive and somatosensory deficits, anxiety-like behavior, and perseverative behavior. The consistent finding of good face validity, together with relevant construct validity, warrant a further evaluation of proteasome inhibition models of Parkinson's disease in pre-clinical research and validation of therapeutic targets.

Metabolically, the brain is a highly active organ that relies almost exclusively on glucose as its energy source. According to the astrocyte-to-neuron lactate shuttle hypothesis, glucose is taken up by astrocytes and converted to lactate, which is then oxidized by neurons. Here we show, using two-photon imaging of a near-infrared 2-deoxyglucose analogue (2DG-IR), that glucose is taken up preferentially by neurons in awake behaving mice. Anaesthesia suppressed neuronal 2DG-IR uptake and sensory stimulation was associated with a sharp increase in neuronal, but not astrocytic, 2DG-IR uptake. Moreover, hexokinase, which catalyses the first enzymatic steps in glycolysis, was highly enriched in neurons compared with astrocytes, in mouse as well as in human cortex. These observations suggest that brain activity and neuronal glucose metabolism are directly linked, and identify the neuron as the principal locus of glucose uptake as visualized by functional brain imaging.

FTY720 (fingolimod, Gilenya™), a structural analog of sphingosine-1-phosphate (S1P), is the first oral drug approved for treatment the relapsing-remitting form of multiple sclerosis (MS), and its efficacy has been related to induced lymphopenia and consequent immunosuppression via modulation of S1P1 receptors (S1P1R). However, due to its lipophilic nature, FTY720 crosses the blood brain barrier (BBB) and could act directly on neural cells. In this study, we investigated the effectiveness of FTY720 as a neuroprotective agent using in vitro and in vivo models of excitotoxic neuronal death and examined if FTY720 exerts a direct action on neurons, or/and an indirect modulation of inflammation-mediated neurodegeneration as a possible mechanism of neuroprotection.Primary neuronal and organotypic cortical cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic cell death (measured by lactate dehydrogenase (LDH) assay or propidium iodide uptake, respectively). The effects of FTY720 treatment (10, 100 and 1,000 nM) on neuronal survival were examined. As an in vivo model of neuronal death and inflammation, we used intracerebroventricular (icv) administration of kainic acid (KA; 0.5 μg/2 μl) in Sprague-Dawley rats. FTY720 was applied icv (1 μg/2 μl), together with KA, plus intraperitoneally (ip; 1 mg/kg) 24 h before, and daily, until sacrifice 3 days after icv. Rats were evaluated for neurological score, neuronal loss in CA3 hippocampal region and activation of microglia at the lesion site. In addition, we tested FTY720 as a modulator of microglia responses using microglial cell cultures activated with lipopolysaccharide (LPS) and its effects in stress signalling pathways using western blotting for p38 and JNK1/2 mitogen-activated protein kinases (MAPKs).FTY720 was able to reduce excitotoxic neuronal death in vitro. Moreover, in vivo repeated FTY720 administration attenuated KA-induced neurodegeneration and microgliosis at the CA3 lesion site. Furthermore, FTY720 negatively modulates p38 MAPK in LPS-activated microglia, whereas it had no effect on JNK1/2 activation.These data support a role for FTY720 as a neuroprotective agent against excitotoxin-induced neuronal death and as a negative modulator of neuroinflammation by targeting the p38 MAPK stress signalling pathway in microglia.

The proinflammatory cytokine osteopontin (OPN) is elevated in the cerebrospinal fluid (CSF) in individuals with HIV-associated neurocognitive disorders (HAND) and remains so in those on suppressive antiretroviral therapy. To understand the pathophysiological significance of elevated OPN in the CNS, we sought to determine the cellular source of this cytokine. As HIV-1 replicates productively in macrophages/microglia, we tested whether these cells are the predominant producers of OPN in the brain. Stringent patient selection criteria, which excluded brain tissues from those with evidence of drug abuse and dependence, were used. Uninfected normal controls, amyotrophic lateral sclerosis (ALS), HIV+ asymptomatic neurocognitive impairment (ANI), and HIV+ mild neurocognitive disorder (MND)/HIV-associated dementia (HAD) groups were included. Double-label immunohistochemistry for CNS cells and OPN was used to quantify OPN expression in astrocytes, macrophages/microglia, and neurons. While resident macrophages/microglia expressed OPN, astrocytes and unexpectedly neurons were also a major source of OPN. OPN levels in ionized Ca(2+)-binding adapter 1 (Iba1)/allograft inflammatory factor-1 (AIF-1)+ microglia in HIV+ ANI and MND/HAD exceeded those of HIV-negative controls and were comparable to expression seen in ALS. Moreover, in neurons, OPN was expressed at the highest levels in the HIV+ ANI group. These findings suggest that while infiltrating HIV-infected macrophages are most likely the initial source of OPN, resident CNS cells become activated and also express this inflammatory cytokine at significant levels. Moreover, as OPN levels are elevated compared to uninfected individuals and increases with the severity of impairment, it appears that the expression of OPN is persistent and sustained within the brain parenchyma in those that progress to HAND.

Neuroinflammation has long been considered a driver of Alzheimer's disease progression. However, experiments developed to explore the interaction between neuroinflammation and Alzheimer's disease (AD) pathology showed a surprising reduction in amyloid beta (Aβ) plaque deposition. We sought to understand this unexpected outcome by examining microglia phenotypes during chronic neuroinflammation.Using an adeno-associated virus vector carrying hIL-1β cDNA, inflammation was induced in one hippocampus of 8-month-old amyloid precursor protein (APP)/PS1 mice for 4 weeks, while the other hemisphere received control injections. Bone marrow chimeras and staining analysis were used to identify the origins and types of immune cells present during sustained inflammation. Arginase 1 (Arg1) and inducible nitric oxide synthase (iNOS) immunoreactivity were used as markers of alternatively activated and classically activated cells, respectively, and changes in cellular uptake of Aβ by Arg1+ or iNOS+ microglia was demonstrated by confocal microscopy. To determine if an anti-inflammatory phenotype was present during neuroinflammation, RNA was extracted on flow-sorted microglia and rt-PCR was performed. Interleukin-4 injection was used to induce alternatively activated cells, whereas a minipump and intrahippocampal cannula was used to deliver an interleukin (IL)-4Rα antibody to block the induction of Arg1+ cells in the setting of sustained IL-1β expression.We observed a robust upregulation of centrally derived Arg1+ microglia present only in the inflamed hemisphere. Furthermore, in the inflamed hemisphere, greater numbers of Arg1+ microglia contained Aβ when compared to iNOS+ microglia. RNA isolated from flow-sorted microglia from the inflamed hemisphere demonstrated elevation of mRNA species consistent with alternative activation as well as neuroprotective genes such as BDNF and IGF1. To explore if Arg1+ microglia mediated plaque reduction, we induced Arg1+ microglia with IL-4 and observed significant plaque clearance. Moreover, when we reduced Arg1+ microglia induction in the context of neuroinflammation using an anti-IL-4Rα antibody delivered via intrahippocampal cannula, we observed a clear correlation between numbers of Arg1+ microglia and plaque reduction.Together, these findings suggest that Arg1+ microglia are involved in Aβ plaque reduction during sustained, IL-1β-dependent neuroinflammation, opening up possible new avenues for immunomodulatory therapy of AD.

Experiencing an adverse childhood and parental neglect is a risk factor for depression in the adult population. Patients with a history of traumatic childhood develop a subtype of depression that is characterized by earlier onset, poor treatment response and more severe symptoms. The long-lasting molecular mechanisms that are engaged during early traumatic events and determine the risk for depression are poorly understood. In this study, we altered adult depression-like behavior in mice by applying juvenile isolation stress. We found that this behavioral phenotype was associated with a reduction in the levels of the deacetylase sirtuin1 (SIRT1) in the brain and in peripheral blood mononuclear cells. Notably, peripheral blood mRNA expression of SIRT1 predicted the extent of behavioral despair only when depression-like behavior was induced by juvenile--but not adult--stress, implicating SIRT1 in the regulation of adult behavior at early ages. Consistent with this hypothesis, pharmacological modulation of SIRT1 during juvenile age altered the depression-like behavior in naive mice. We also performed a pilot study in humans, in which the blood levels of SIRT1 correlated significantly with the severity of symptoms in major depression patients, especially in those who received less parental care during childhood. On the basis of these novel findings, we propose the involvement of SIRT1 in the long-term consequences of adverse childhood experiences.

Mutations in the charged multivesicular body protein 2B (CHMP2B) cause frontotemporal dementia (FTD). We report that mice which express FTD-causative mutant CHMP2B at physiological levels develop a novel lysosomal storage pathology characterised by large neuronal autofluorescent aggregates. The aggregates are an early and progressive pathology that occur at 3 months of age and increase in both size and number over time. These autofluorescent aggregates are not observed in mice expressing wild-type CHMP2B, or in non-transgenic controls, indicating that they are a specific pathology caused by mutant CHMP2B. Ultrastructural analysis and immuno- gold labelling confirmed that they are derived from the endolysosomal system. Consistent with these findings, CHMP2B mutation patient brains contain morphologically similar autofluorescent aggregates. These aggregates occur significantly more frequently in human CHMP2B mutation brain than in neurodegenerative disease or age-matched control brains. These data suggest that lysosomal storage pathology is the major neuronal pathology in FTD caused by CHMP2B mutation. Recent evidence suggests that two other genes associated with FTD, GRN and TMEM106B are important for lysosomal function. Our identification of lysosomal storage pathology in FTD caused by CHMP2B mutation now provides evidence that endolysosomal dysfunction is a major degenerative pathway in FTD.

A GABAergic component has been identified in the projection from the inferior colliculus (IC) to the medial geniculate body (MG) in cats and rats. We sought to determine if this GABAergic pathway exists in guinea pig, a species widely used in auditory research. The guinea pig IC contains GABAergic cells, but their relative abundance in the IC and their relative contributions to tectothalamic projections are unknown. We identified GABAergic cells with immunochemistry for glutamic acid decarboxylase (GAD) and determined that ~21% of IC neurons are GABAergic. We then combined retrograde tracing with GAD immunohistochemistry to identify the GABAergic tectothalamic projection. Large injections of Fast Blue, red fluorescent beads or FluoroGold were deposited to include all subdivisions of the MG. The results demonstrate a GABAergic pathway from each IC subdivision to the ipsilateral MG. GABAergic cells constitute ~22% of this ipsilateral pathway. In addition, each subdivision of the IC had a GABAergic projection to the contralateral MG. Measured by number of tectothalamic cells, the contralateral projection is about 10% of the size of the ipsilateral projection. GABAergic cells constitute about 20% of the contralateral projection. In summary, the results demonstrate a tectothalamic projection in guinea pigs that originates in part from GABAergic cells that project ipsilaterally or contralaterally to the MG. The results show similarities to both rats and cats, and carry implications for the role of GABAergic tectothalamic projections vis-à-vis the presence (in cats) or near absence (in rats and guinea pigs) of GABAergic interneurons in the MG.