Hope for vascular cognitive impairment: Ac-YVAD-cmk as a novel treatment against white matter rarefaction.

Vascular cognitive impairment (VCI) is the second leading cause of dementia with limited treatment options, characterised by cerebral hypoperfusion-induced white matter rarefaction (WMR). Subcortical VCI is the most common form of VCI, but the underlying reasons for region susceptibility remain elusive. Recent studies employing the bilateral cortical artery stenosis (BCAS) method demonstrate that various inflammasomes regulate white matter injury and blood-brain barrier dysfunction but whether caspase-1 inhibition will be beneficial remains unclear. To address this, we performed BCAS on C57/BL6 mice to study the effects of Ac-YVAD-cmk, a caspase-1 inhibitor, on the subcortical and cortical regions. Cerebral blood flow (CBF), WMR, neuroinflammation and the expression of tight junction-related proteins associated with blood-brain barrier integrity were assessed 15 days post BCAS. We observed that Ac-YVAD-cmk restored CBF, attenuated BCAS-induced WMR and restored subcortical myelin expression. Within the subcortical region, BCAS activated the NLRP3/caspase-1/interleukin-1beta axis only within the subcortical region, which was attenuated by Ac-YVAD-cmk. Although we observed that BCAS induced significant increases in VCAM-1 expression in both brain regions that were attenuated with Ac-YVAD-cmk, only ZO-1 and occludin were observed to be significantly altered in the subcortical region. Here we show that caspase-1 may contribute to subcortical regional susceptibility in a mouse model of VCI. In addition, our results support further investigations into the potential of Ac-YVAD-cmk as a novel treatment strategy against subcortical VCI and other conditions exhibiting cerebral hypoperfusion-induced WMR.

Chronic treatment with baicalein alleviates behavioural disorders and improves cerebral blood flow via reverting metabolic abnormalities in a J20 transgenic mouse model of Alzheimer's disease.

Baicalein (BE) has both antioxidant and anti-inflammatory effects. It has also been reported able to improve cerebral blood circulation in brain ischemic injury. However, its chronic efficacy and metabolomics in Alzheimer's disease (AD) remain unknown. In this study, BE at 80 mg/kg was administrated through the oral route in J20 AD transgenic mice aged from aged 4 months to aged 10 months. Metabolic- and neurobehavioural phenotyping was done before and after 6 months' treatment to evaluate the drug efficacy and the relevant mechanisms. Meanwhile, molecular docking was used to study the binding affinity of BE and poly (ADP-ribose) polymerase-1 (PARP-1) which is related to neuronal injury. The open field test showed that BE could suppress hyperactivity in J20 mice and increase the frequency of the target quadrant crossing in the Morris Water Maze test. More importantly, BE restored cerebral blood flow back to the normal level after the chronic treatment. A 1H NMR-based metabolomics study showed that BE treatment could restore the tricarboxylic acid cycle in plasma. And such a treatment could suppress oxidative stress, inhibit neuroinflammation, alleviate mitochondrial dysfunction, improve neurotransmission, and restore amino homeostasis via starch and sucrose metabolism and glycolipid metabolism in the cortex and hippocampus, which could affect the behavioural and cerebral blood flow. These findings showed that BE is a potential therapeutic agent for AD.

Effect of fingolimod on oligodendrocyte maturation under prolonged cerebral hypoperfusion.

Oligodendrocytes (OLGs) support neuronal system and have crucial roles for brain homeostasis. As the renewal and regeneration of OLGs derived from oligodendrocyte precursor cells (OPCs) are inhibited by various pathological conditions, the restoration of impaired oligodendrogenesis is a therapeutic strategy for OLG-related diseases such as subcortical ischemic vascular dementia (SIVD). Fingolimod (FTY720), a drug for multiple sclerosis, is reported to elicit a cytoprotective effect on OPCs in vitro. However, the effects of fingolimod against ischemia-induced suppression of OPC differentiation remain unknown. Hence, the purpose of this study was to investigate the effectiveness of fingolimod against ischemia-induced suppression of oligodendrogenesis. For the in vitro experiments, primary rat cultured OPCs were incubated with a non-lethal concentration of CoCl2 to induce chemical hypoxic conditions and were treated with or without fingolimod-phosphate. We found that low concentration fingolimod-phosphate directly rescued ischemia-induced suppression of OPC differentiation via the phosphoinositide 3-kinase-Akt pathway. For the in vivo experiments, we used a mouse model of SIVD generated by bilateral common carotid artery stenosis. On day 28 after surgery, fingolimod ameliorated ischemia-induced demyelination and promoted oligodendrogenesis under prolonged cerebral hypoperfusion. The present study demonstrates that fingolimod can promote oligodendrogenesis under ischemic conditions and may be a therapeutic candidate for SIVD.

Cerebral transcriptome analysis reveals age-dependent progression of neuroinflammation in P301S mutant tau transgenic male mice.

Aggregation of the microtubule-associated protein, tau, can lead to neurofibrillary tangle formation in neurons and glia which is the hallmark of tauopathy. The cellular damage induced by the formation of neurofibrillary tangles leads to neuroinflammation and consecutive neuronal death. However, detailed observation of transcriptomic changes under tauopathy together with the comparison of age-dependent progression of neuroinflammatory gene expressions mediated by tau overexpression is required. Employing RNA sequencing on PS19 transgenic mice that overexpress human mutant tau harboring the P301S mutation, we have examined the effects of age-dependent tau overexpression on transcriptomic changes of immune and inflammatory responses in the cerebral cortex. Compared to age-matched wild type control, P301S transgenic mice exhibit significant transcriptomic alterations. We have observed age-dependent neuroinflammatory gene expression changes in both wild type and P301S transgenic mice where tau overexpression further promoted the expression of neuroinflammatory genes in 10-month old P301S transgenic mice. Moreover, functional gene network analyses (gene ontology and pathway enrichment) and prospective target protein interactions predicted the potential involvement of multiple immune and inflammatory pathways that may contribute to tau-mediated neuronal pathology. Our current study on P301S transgenic mice model revealed for the first time, the differences of gene expression patterns in both early and late stage of tau pathology in cerebral cortex. Our analyses also revealed that tau overexpression alone induces multiple inflammatory and immune transcriptomic changes and may provide a roadmap to elucidate the targets of anti-inflammatory therapeutic strategy focused on tau pathology and related neurodegenerative diseases.

Epigenetic regulation of inflammation in stroke.

Despite extensive research, treatments for clinical stroke are still limited only to the administration of tissue plasminogen activator and the recent introduction of mechanical thrombectomy, which can be used in only a limited proportion of patients due to time constraints. A plethora of inflammatory events occur during stroke, arising in part due to the body's immune response to brain injury. Neuroinflammation contributes significantly to neuronal cell death and the development of functional impairment and death in stroke patients. Therefore, elucidating the molecular and cellular mechanisms underlying inflammatory damage following stroke injury will be essential for the development of useful therapies. Research findings increasingly point to the likelihood that epigenetic mechanisms play a role in the pathophysiology of stroke. Epigenetics involves the differential regulation of gene expression, including those involved in brain inflammation and remodelling after stroke. Hence, it is conceivable that epigenetic mechanisms may contribute to differential interindividual vulnerability and injury responses to cerebral ischaemia. In this review, we summarize recent findings on the emerging role of epigenetics in the regulation of neuroinflammation in stroke. We also discuss potential epigenetic targets that may be assessed for the development of stroke therapies.

Evidence that NF-κB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke.

Multi-protein complexes, termed "inflammasomes," are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1ß and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.

The effects of MLC901 on tau phosphorylation.

Tauopathies are neurodegenerative diseases that are characterized by the presence of hyperphosphorylated tau-containing neurofibrillary tangles (NFTs) in the brain and include Alzheimer's disease and frontotemporal dementia, which lack effective disease-modifying treatments. The presence of NFTs is known to correlate with cognition impairment, suggesting that targeting tau hyperphosphorylation may be therapeutically effective. MLC901 is a herbal formulation that is currently used in poststroke recovery and consists of nine herbal components. Previously, several components of MLC901 have been shown to have an effect on tau phosphorylation, but it remains unknown whether MLC901 itself has the same effect. The objective of this study was to assess the effects of MLC901 on ameliorating tau phosphorylation at epitopes associated with NFT formation. A stably transfected cell culture model expressing tau harboring the P301S mutation was generated and treated with various concentrations of MLC901 across different time points. Tau phosphorylation profiles and protein levels of enzymes associated with tau phosphorylation were assessed using western blotting. One-way analysis of variance with Bonferroni post-hoc analysis showed that MLC901 significantly reduced tau phosphorylation at epitopes recognized by the AT8, AT270, and PHF-13 antibodies. MLC901 also induced a significant increase in the s9 phosphorylation of glycogen synthase kinase 3ß and a concurrent decrease in the activation of cyclin-dependent kinase 5, as measured by a significant decrease in the levels of p35/cyclin-dependent kinase 5. Our results provide supporting evidence to further study the effects of MLC901 on tau pathology and cognition using mouse models of tauopathy.

Increased phosphorylation of collapsin response mediator protein-2 at Thr514 correlates with ß-amyloid burden and synaptic deficits in Lewy body dementias.

Collapsin response mediator protein-2 (CRMP2) regulates axonal growth cone extension, and increased CRMP2 phosphorylation may lead to axonal degeneration. Axonal and synaptic pathology is an important feature of Lewy body dementias (LBD), but the state of CRMP2 phosphorylation (pCRMP2) as well as its correlations with markers of neurodegeneration have not been studied in these dementias. Hence, we measured CRMP2 phosphorylation at Thr509, Thr514 and Ser522, as well as markers of ß-amyloid (Aß), tau-phosphorylation, α-synuclein and synaptic function in the postmortem neocortex of a longitudinally assessed cohort of LBD patients characterized by low (Parkinson's disease dementia, PDD) and high (dementia with Lewy bodies, DLB) burden of Alzheimer type pathology. We found specific increases of pCRMP2 at Thr514 in DLB, but not PDD. The increased CRMP2 phosphorylation correlated with fibrillogenic Aß as well as with losses of markers for axon regeneration (ß-III-tubulin) and synaptic integrity (synaptophysin) in LBD. In contrast, pCRMP2 alterations did not correlate with tau-phosphorylation or α-synuclein, and also appear unrelated to immunoreactivities of putative upstream kinases glycogen synthase kinase 3ß and cyclin-dependent kinase 5, as well as to protein phosphatase 2A. In conclusion, increased pCRMP2 may underlie the axonal pathology of DLB, and may be a novel therapeutic target. However, antecedent signaling events as well as the nature of pCRMP2 association with Aß and other neuropathologic markers require further study.

Lessons from two prevalent amyloidoses-what amylin and Aß have in common.

The amyloidogenic peptide Aß plays a key role in Alzheimer's disease (AD) forming insoluble aggregates in the brain. The peptide shares its amyloidogenic properties with amylin that forms aggregates in the pancreas of patients with Type 2 Diabetes mellitus (T2DM). While epidemiological studies establish a link between these two diseases, it is becoming increasingly clear that they also share biochemical features suggesting common pathogenic mechanisms. We discuss commonalities as to how Aß and amylin deregulate the cellular proteome, how they impair mitochondrial functions, to which receptors they bind, aspects of their clearance and how therapeutic strategies exploit the commonalities between Aß and amylin. We conclude that research into these two molecules is mutually beneficial for the treatment of AD and T2DM.

Alzheimer's disease, oestrogen and mitochondria: an ambiguous relationship.

Hormonal deficit in post-menopausal women has been proposed to be one risk factor in Alzheimer's disease (AD) since two thirds of AD patients are women. However, large treatment trials showed negative effects of long-term treatment with oestrogens in older women. Thus, oestrogen treatment after menopause is still under debate, and several hypotheses trying to explain the failure in outcome are under discussion. Concurrently, it was shown that amyloid-beta (Aß) peptide, the main constituent of senile plaques, as well as abnormally hyperphosphorylated tau protein, the main component of neurofibrillary tangles, can modulate the level of neurosteroids which notably represent neuroactive steroids synthetized within the nervous system, independently of peripheral endocrine glands. In this review, we summarize the role of neurosteroids especially that of oestrogen in AD and discuss their potentially neuroprotective effects with specific regard to the role of oestrogens on the maintenance and function of mitochondria, important organelles which are highly vulnerable to Aß- and tau-induced toxicity. We also discuss the role of Aß-binding alcohol dehydrogenase (ABAD), a mitochondrial enzyme able to bind Aß peptide thereby modifying mitochondrial function as well as oestradiol levels suggesting possible modes of interaction between the three, and the potential therapeutic implication of inhibiting Aß-ABAD interaction.

Role of hippocalcin in mediating Aß toxicity.

Alzheimer's disease (AD) is the most common cause of dementia, and amyloid-ß (Aß) plaques and tau-containing tangles are its histopathological hallmark lesions. These do not occur at random; rather, the neurodegenerative process is stereotyped in that it is initiated in the entorhinal cortex and hippocampal formation. Interestingly, it is the latter brain area where the calcium-sensing enzyme hippocalcin is highly expressed. Because calcium deregulation is a well-established pathomechanism in AD, we aimed to address the putative role of hippocalcin in human AD brain and transgenic mouse models. We found that hippocalcin levels are increased in human AD brain and in Aß plaque-forming APP23 transgenic mice compared to controls. To determine the role of hippocalcin in Aß toxicity, we treated primary cultures derived from hippocalcin knockout (HC KO) mice with Aß and found them to be more susceptible to Aß toxicity than controls. Likewise, treatment with either thapsigargin or ionomycin, both known to deregulate intracellular calcium levels, caused an increased toxicity in hippocampal neurons from HC KO mice compared to wild-type. We found further that mitochondrial complex I activity increased from 3 to 6months in hippocampal mitochondria from wild-type and HC KO mice, but that the latter exhibited a significantly stronger aging phenotype than wild-type. Aß treatment induced significant toxicity on hippocampal mitochondria from HC KO mice already at 3months of age, while wild-type mitochondria were spared. Our data suggest that hippocalcin has a neuroprotective role in AD, presenting it as a putative biomarker.

Inhibition of the mitochondrial enzyme ABAD restores the amyloid-ß-mediated deregulation of estradiol.

Alzheimer's disease (AD) is a conformational disease that is characterized by amyloid-ß (Aß) deposition in the brain. Aß exerts its toxicity in part by receptor-mediated interactions that cause down-stream protein misfolding and aggregation, as well as mitochondrial dysfunction. Recent reports indicate that Aß may also interact directly with intracellular proteins such as the mitochondrial enzyme ABAD (Aß binding alcohol dehydrogenase) in executing its toxic effects. Mitochondrial dysfunction occurs early in AD, and Aß's toxicity is in part mediated by inhibition of ABAD as shown previously with an ABAD decoy peptide. Here, we employed AG18051, a novel small ABAD-specific compound inhibitor, to investigate the role of ABAD in Aß toxicity. Using SH-SY5Y neuroblastoma cells, we found that AG18051 partially blocked the Aß-ABAD interaction in a pull-down assay while it also prevented the Aß42-induced down-regulation of ABAD activity, as measured by levels of estradiol, a known hormone and product of ABAD activity. Furthermore, AG18051 is protective against Aß42 toxicity, as measured by LDH release and MTT absorbance. Specifically, AG18051 reduced Aß42-induced impairment of mitochondrial respiration and oxidative stress as shown by reduced ROS (reactive oxygen species) levels. Guided by our previous finding of shared aspects of the toxicity of Aß and human amylin (HA), with the latter forming aggregates in Type 2 diabetes mellitus (T2DM) pancreas, we determined whether AG18051 would also confer protection from HA toxicity. We found that the inhibitor conferred only partial protection from HA toxicity indicating distinct pathomechanisms of the two amyloidogenic agents. Taken together, our results present the inhibition of ABAD by compounds such as AG18051 as a promising therapeutic strategy for the prevention and treatment of AD, and suggest levels of estradiol as a suitable read-out.

Dissecting toxicity of tau and beta-amyloid.

BACKGROUND: How beta-amyloid (Abeta) and tau exert toxicity in Alzheimer's disease is only partly understood. Major questions include (1) which aggregation state of Abeta confers toxicity, (2) do amyloidogenic proteins have similar mechanisms of toxicity, and (3) does soluble tau interfere with cellular functions? METHODS: To determine Abeta toxicity in P301L mutant tau transgenic mice, mitochondrial function was assessed after insult with monomeric, oligomeric and fibrillar Abeta. Amylin and Abeta toxicity were compared in cortical and hippocampal long-term cultures. To determine tau toxicity, K369I mutant tau mice were established as a model of frontotemporal dementia, analyzed biochemically and compared with human diseased brain. RESULTS: Oligomeric and fibrillar Abeta42 were both toxic, although to different degrees. Human amylin shared toxicity with Abeta42, an effect not observed for nonamyloidogenic rat amylin. Clinical features of K369I tau mice were caused by aberrant interaction of phosphorylated tau with JIP1, a component of the kinesin transport machinery. CONCLUSION: Our data support the notion of a synergistic action of tau and Abeta pathology on mitochondria. A specific conformation of Abeta42 and human amylin determines toxicity. Finally, trapping of JIP1 by phosphorylated tau in the neuronal soma emerges as a fundamental pathomechanism in neurodegeneration.

Abeta and human amylin share a common toxicity pathway via mitochondrial dysfunction.

Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) are leading causes of morbidity and mortality in the elderly. Both diseases are characterized by amyloid deposition in target tissues: aggregation of amylin in T2DM is associated with loss of insulin-secreting beta-cells, while amyloid beta (A beta) aggregation in AD brain is associated with neuronal loss. Here, we used quantitative iTRAQ proteomics as a discovery tool to show that both A beta and human amylin (HA) deregulate identical proteins, a quarter of which are mitochondrial, supporting the notion that mitochondrial dysfunction is a common target in these two amyloidoses. A functional validation revealed that mitochondrial complex IV activity was significantly reduced after treatment with either HA or A beta, as was mitochondrial respiration. In comparison, complex I activity was reduced only after treatment with HA. A beta and HA, but not the non-amyloidogenic rat amylin, induced significant increases in the generation of ROS. Co-incubation of HA and A beta did not produce an augmented effect in ROS production, again suggesting common toxicity mechanisms. In conclusion, our data suggest that A beta and HA both exert toxicity, at least in part, via mitochondrial dysfunction, thus restoring their function may be beneficial for both AD and T2DM.

Oxidative stress increases levels of endogenous amyloid-beta peptides secreted from primary chick brain neurons.

Oxidative damage is associated with Alzheimer's disease and mild cognitive impairment, but its relationship to the development of neuropathological lesions involving accumulation of amyloid-beta (Abeta) peptides and hyperphosphorylated tau protein remains poorly understood. We show that inducing oxidative stress in primary chick brain neurons by exposure to sublethal doses of H(2)O(2 )increases levels of total secreted endogenous Abeta by 2.4-fold after 20 h. This occurs in the absence of changes to intracellular amyloid precursor protein or tau protein levels, while heat-shock protein 90 is elevated 2.5-fold. These results are consistent with the hypothesis that aging-associated oxidative stress contributes to increasing Abeta generation and up-regulation of molecular chaperones in Alzheimer's disease.

Human but not rat amylin shares neurotoxic properties with Abeta42 in long-term hippocampal and cortical cultures.

Type 2 diabetes mellitus (DM) and Alzheimer's disease (AD) share epidemiological and biochemical features. Both are characterized by insoluble protein aggregates with a fibrillar conformation--amylin in Type 2 DM pancreatic islets, and Abeta in AD brain. To determine whether amylin shares neurotoxic properties with Abeta, we incubated hippocampal and cortical neurons with Abeta42 and human amylin. Different from non-amyloidogenic rat amylin, both caused a dose-, time- and cell type-specific neurotoxicity supporting the notion of a similar toxic mechanism. Depending on the cell type, this finding is also supported by co-incubation of human amylin and Abeta.

Functional genomics dissects pathomechanisms in tauopathies: mitosis failure and unfolded protein response.

BACKGROUND: Alzheimer's disease (AD) is characterized by beta-amyloid (Abeta) peptide-containing plaques and tau-containing neurofibrillary tangles. By intracerebral injection of Abeta(42), both pathologies have been combined in P301L tau mutant mice. Furthermore, in cell culture, Abeta(42) induces tau aggregation. While both Abeta(42) and mutant tau cause neuronal dysfunction, their modes of action are only vaguely understood. METHODS: To determine which processes are disrupted by Abeta(42) and/or P301L mutant tau, we used transcriptomic and proteomic techniques followed by functional validation and analysis of human AD tissue. RESULTS: Our transcriptomic study in the SH-SY5Y cell culture system revealed that Abeta(42) and P301L tau expression independently affect genes controlling the cell cycle and cell proliferation. Proteomics applied to Abeta(42)-treated P301L tau-expressing SH-SY5Y cells and the amygdala of Abeta(42)-injected P301L transgenic mice revealed that a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. Among the proteins identified was valosin-containing protein (VCP), a component of the quality control system during endoplasmic reticulum stress. Mutations in VCP have recently been linked to frontotemporal dementia. CONCLUSION: Our data support the mitosis failure hypothesis that claims that aberrant cell cycle reentry of postmitotic neurons induces apoptosis. Furthermore, our data underline a role of Abeta(42) in the stress response associated with protein folding.

Altered levels of PP2A regulatory B/PR55 isoforms indicate role in neuronal differentiation.

The ubiquitously expressed serine/threonine-specific protein phosphatase 2A (PP2A) is prominent in brain where it serves a wide range of functions under both physiological and pathological conditions. PP2A holoenzymes are composed of a catalytic subunit and a tightly complexed scaffolding subunit. This core enzyme associates with regulatory subunits of the B/PR55, B'/PR56/PR61, B''/PR72 and B'''/PR93/PR110 families. We previously determined distribution and expression levels of the four members of the B/PR55 family in brain, as dysregulation of this subunit family has been specifically implicated in neurodegenerative disorders including Alzheimer's disease. In the present study, we used cell lines widely used in neuroscience research to determine levels of the four PR55 isoforms by qRT-PCR under different experimental conditions. We show that PR55alpha mRNA levels are highest in both HEK293 cells and SH-SY5Y neuroblastoma cells whereas PR55beta levels are lowest. Stepwise neuronal differentiation of SH-SY5Y cells causes the selective upregulation of PR55beta, and to some extent PR55gamma and PR55delta, but not PR55alpha mRNAs. In agreement with the qRT-PCR analysis, neuronal differentiation does not alter PR55alpha protein levels, whereas interestingly, PR55beta and PR55gamma protein levels are reduced when compared to undifferentiated cells. Our data point at specific roles for distinct regulatory B/PR55 subunits of PP2A in neuron-like cells with PR55alpha being the major isoform.