Julie Vallortigara

Methylglyoxal Evokes Pain by Stimulating TRPA1

Diabetic neuropathy is a severe complication of long-standing diabetes and one of the major etiologies of neuropathic pain. Diabetes is associated with an increased formation of reactive oxygen species and the electrophilic dicarbonyl compound methylglyoxal (MG). Here we show that MG stimulates heterologously expressed TRPA1 in CHO cells and natively expressed TRPA1 in MDCK cells and DRG neurons. MG evokes [Ca2+]i-responses in TRPA1 expressing DRG neurons but is without effect in neurons cultured from Trpa1−/− mice. Consistent with a direct, intracellular action, we show that methylglyoxal is significantly more potent as a TRPA1 agonist when applied to the intracellular face of excised membrane patches than to intact cells. Local intraplantar administration of MG evokes a pain response in Trpa1+/+ but not in Trpa1−/− mice. Furthermore, persistently increased MG levels achieved by two weeks pharmacological inhibition of glyoxalase-1 (GLO-1), the rate-limiting enzyme responsible for detoxification of MG, evokes a progressive and marked thermal (cold and heat) and mechanical hypersensitivity in wildtype but not in Trpa1−/− mice. Our results thus demonstrate that TRPA1 is required both for the acute pain response evoked by topical MG and for the long-lasting pronociceptive effects associated with elevated MG in vivo. In contrast to our observations in DRG neurons, MG evokes indistinguishable [Ca2+]i-responses in pancreatic β-cells cultured from Trpa1+/+ and Trpa1−/− mice. In vivo, the TRPA1 antagonist HC030031 impairs glucose clearance in the glucose tolerance test both in Trpa1+/+ and Trpa1−/− mice, indicating a non-TRPA1 mediated effect and suggesting that results obtained with this compound should be interpreted with caution. Our results show that TRPA1 is the principal target for MG in sensory neurons but not in pancreatic β-cells and that activation of TRPA1 by MG produces a painful neuropathy with the behavioral hallmarks of diabetic neuropathy.

Assessment of ZnT3 and PSD95 protein levels in Lewy body dementias and Alzheimer's disease: association with cognitive impairment

The loss of zinc transporter 3 (ZnT3) has been implicated in age-related cognitive decline in mice, and the protein has been associated with plaques. We investigated the levels of ZnT3 and postsynaptic density protein 95 (PSD95), a marker of the postsynaptic terminal, in people with Parkinsons disease dementia (PDD, nxa0= 31), dementia with Lewy bodies (DLB, nxa0= 44), Alzheimers disease (AD, nxa0= 16), and controls (nxa0= 24), using semiquantitative western blotting and immunohistochemistry in 3 cortical regions. Standardized cognitive assessments during life and semiquantitative scoring of amyloid β (Aβ), tau, and α-synuclein at postmortem were used to investigate the relationship between ZnT3 and PSD95, cognition and pathology. Associations were observed between ZnT3 and PSD95 levels in prefrontal cortex and cognitive impairment (pxa0= 0.001 and pxa0= 0.002, respectively) and between ZnT3 levels in the parietal cortex and cognitive impairment (pxa0= 0.036). Associations were also seen between ZnT3 levels in cingulate cortex and severity of Aβ (pxa0= 0.003) and tau (pxa0= 0.011) pathologies. DLB and PDD were characterized by significant reductions of PSD95 (p < 0.05) and ZnT3 (p < 0.001) in prefrontal cortex compared with controls and AD. PSD95 levels in the parietal cortex were found to be decreased in AD cases compared with controls (pxa0= 0.02) and PDD (pxa0= 0.005). This study has identified Zn(2+) modulation as a possible novel target for the treatment of cognitive impairment in DLB and PDD and the potential for synaptic proteins to be used as a biomarker for the differentiation of DLB and PDD from AD.

Depression and synaptic zinc regulation in Alzheimer disease, dementia with lewy bodies, and Parkinson disease dementia.

OBJECTIVEnDepression is a common symptom in dementia with Lewy bodies (DLB), Parkinson disease dementia (PDD), and Alzheimer disease (AD), yet its molecular basis remains unclear and current antidepressants do not appear to be effective. Cerebral zinc has been implicated in depression and synaptic dysfunction. We investigated the relationship between synaptic zinc regulation (for which zinc transporter 3 [ZnT3] is responsible) and depression in a large clinicopathologic study.nnnMETHODSnWe examined brains from people with PDD (Nxa0= 29), DLB (Nxa0= 27), and AD (Nxa0= 15) and comparison subjects without depression or dementia (Nxa0= 24). Individuals were categorized according to the presence and severity of depression (on a scale of 0-3) based on standardized assessments during life (principally Neuropsychiatric Inventory). Western blotting was used to determine ZnT3 levels in Brodmann area 9 (BA9), and regression analysis was used to determine the relationship between ZnT3 and depression.nnnRESULTSnReductions in ZnT3 in BA9 were significantly associated with elevated depression scores in the study cohort (βxa0=xa0-0.351, dfxa0= 93, txa0=xa0-3.318 pxa0= 0.0004). This association remained when only individuals with DLB, PDD, and no dementia or depression were examined (βxa0=xa0-0.347, dfxa0= 78, txa0=xa0-3.271, pxa0= 0.002) or only individuals with AD and no dementia or depression were examined (βxa0=xa0-0.433, dfxa0= 37, txa0=xa0-2.924, pxa0= 0.006).nnnCONCLUSIONnAlthough decreased zinc levels have been implicated in the genesis of depression in animal models and in major depressive disorder in humans, this study provides the first evidence of a role for zinc in depression in people with dementia and highlights zinc metabolism as a therapeutic target.

Decreased Levels of VAMP2 and Monomeric Alpha-Synuclein Correlate with Duration of Dementia

Alpha-synuclein (α-syn) aggregations are the key pathological hallmark of dementia with Lewy bodies (DLB) and Parkinsons disease dementia (PDD), but are also frequently present in Alzheimers disease (AD). Much remains unknown about the role of α-syn in the synapse and the wider role of synaptic dysfunction in these dementias. Changes in concentrations of key SNAP (Soluble N-ethylmaleimide Sensitive Factor Attachment Protein) Receptor (SNARE) proteins as a consequence of alterations in the aggregation state of α-syn may contribute to synaptic dysfunction in patients with DLB, PDD, and AD and result in impaired cognition. We have studied a large cohort (nu200a=u200a130) of autopsy confirmed DLB, PDD, AD, and control brains. Using semi-quantitative western blotting, we have demonstrated significant changes across the diagnostic groups of DLB, PDD, and AD in the SNARE and vesicle proteins syntaxin, Munc18, VAMP2, and monomeric α-syn in the prefrontal cortex, with a significant reduction of Munc18 in AD patients (pu200a< u200a0.001). This correlated to the final MMSE score before death (pu200a=u200a0.016). We also identified a significant negative correlation between the duration of dementia and the levels of the binding partners VAMP2 (pu200a=u200a0.0004) and monomeric α-syn (pu200a=u200a0.0002). Our findings may indicate that an upregulation of SNARE complex related proteins occurs in the early stages of disease as an attempt at compensating for failing synapses, prior to widespread deposition of pathological α-syn.

Dynamin1 concentration in the prefrontal cortex is associated with cognitive impairment in Lewy body dementia

Dementia with Lewy Bodies (DLB) and Parkinson’s Disease Dementia (PDD) together, represent the second most common cause of dementia, after Alzheimer’s disease (AD). The synaptic dysfunctions underlying the cognitive decline and psychiatric symptoms observed throughout the development of PDD and DLB are still under investigation. In this study we examined the expression level of Dynamin1 and phospho-CaMKII, key proteins of endocytosis and synaptic plasticity respectively, as potential markers of molecular processes specifically deregulated with DLB and/or PDD. In order to measure the levels of these proteins, we isolated grey matter from post-mortem prefrontal cortex area (BA9), anterior cingulated gyrus (BA24) and parietal cortex (BA40) from DLB and PDD patients in comparison to age-matched controls and a group of AD cases. Clinical and pathological data available included the MMSE score, neuropsychiatric history, and semi-quantitative scores for AD pathology (plaques - tangles) and for α-synuclein (Lewy bodies). Changes in the expression of the synaptic markers, and correlates with neuropathological features and cognitive decline were predominantly found in the prefrontal cortex. On one hand, levels of Dynamin1 were significantly reduced, and correlated with a higher rate of cognitive decline observed in cases from three dementia groups. On the other hand, the fraction of phospho-CaMKII was decreased, and correlated with a high score of plaques and tangles in BA9. Interestingly, the correlation between the rate of cognitive decline and the level of Dynamin1 remained when the analysis was restricted to the PDD and DLB cases, highlighting an association of Dynamin1 with cognitive decline in people with Lewy Body dementia.

Reduction of RPT6/S8 (a Proteasome Component) and Proteasome Activity in the Cortex is Associated with Cognitive Impairment in Lewy Body Dementia

Lewy body dementia is the second most common neurodegenerative dementia and is pathologically characterized by α-synuclein positive cytoplasmic inclusions, with varying amounts of amyloid-β (Aβ) and hyperphosphorylated tau (tau) aggregates in addition to synaptic loss. A dysfunctional ubiquitin proteasome system (UPS), the major proteolytic pathway responsible for the clearance of short lived proteins, may be a mediating factor of disease progression and of the development of α-synuclein aggregates. In the present study, protein expression of a key component of the UPS, the RPT6 subunit of the 19S regulatory complex was determined. Furthermore, the main proteolytic-like (chymotrypsin- and PGPH-) activities have also been analyzed. The middle frontal (Brodmann, BA9), inferior parietal (BA40), and anterior cingulate (BA24) gyrus’ cortex were selected as regions of interest from Parkinson’s disease dementia (PDD, nu200a=u200a31), dementia with Lewy bodies (DLB, nu200a=u200a44), Alzheimer’s disease (AD, nu200a=u200a16), and control (nu200a=u200a24) brains. Clinical and pathological data available included the MMSE score. DLB, PDD, and AD were characterized by significant reductions of RPT6 (one-way ANOVA, pu200a<u200a0.001; Bonferroni post hoc test) in prefrontal cortex and parietal cortex compared with controls. Strong associations were observed between RPT6 levels in prefrontal, parietal cortex, and anterior cingulate gyrus and cognitive impairment (pu200a=u200a0.001, pu200a=u200a0.001, and pu200a=u200a0.008, respectively). These findings highlight the involvement of the UPS in Lewy body dementia and indicate that targeting the UPS may have the potential to slow down or reduce the progression of cognitive impairment in DLB and PDD.

Dynamin protein in stroke and vascular dementia

Damage to sub-cortical white matter is a key substrate of vascular dementia (VaD) leading to deficits in executive function and cognitive processing speed. Dynamin1 is a 100 kDa protein, accounting for 0.4% of the total brain protein, and has a central role in many intracellular processes such as synaptic vesicle trafficking and recycling. In this study, we examined the status of Dynamin1 in the white matter from frontal cortex area. In order to measure the levels of Dynamin1, we isolated cortical white matter from a total of 34 post-mortem brains derived from controls (N=11), mixed Alzheimers disease (AD) and VaD (N=8), VaD (N=7), and stroke no dementia (SND, N=8) subjects. A commercial ELISA kit was then used to determine the level of Dynamin1. In comparison to controls, Dynamin1 was elevated in patients SND (+400%) and reduced in patients with mixed VaD (-50%). Furthermore, levels of Dynamin1 were significantly associated with preserved cognition as indicated by the MMSE and CAMCOG and upregulation of vesicular glutamate transporter 1. This work indicates that Dynamin1 is associated with both preserved cognition and regenerative responses in older people with cerebrovascular disease and may represent a novel treatment target.

RPT6, 20S α-6 AND α-3 PROTEASOME SUBUNIT LEVELS AND ASSOCIATION WITH COGNITIVE DECLINE IN ALZHEIMER’S DISEASE AND LEWY BODY DEMENTIAS

roles and is linked to several cell death-related signaling pathways in ischemia. Methods: DAPK1-NR2B, DAPK1-DANGER, DAPK1-p53, and DAPK1-Tau are currently known pathways in strokeinduced cell death, we block these cascades in an acute treatment to see whether it will reduce damage. Results: Block DAPK1-Tau interaction can effectively reduce neuronal loss. Conclusions:Disrupting DAPK1-relevant cell death pathway could be considered as a promising therapeutic approach in stroke.

Proteasome and p62 protein impairment of the frontal cortex and anterior cingulate in Lewy body dementias

croglia and astrocytes were isolated from brain tissue of non-demented control and Alzheimer’s disease cases, obtained at autopsy with short pot mortem delay time (average 6 hrs) through cooperation with the Netherlands Brain Bank. To study the uptake pathway of Aß, cells were plated and preincubated for 1 hour with different inhibitors, before exposure to fluorescence labelled (FAM) oligomeric and fibrillar forms of synthetic Aß (10 uM). After 18 hours of incubation, the number of amyloid positive cells was quantified by flow cytometry. Inhibitors of various pathways (to be) tested, include Cytochalasin B (inhibits actin polymerization; general endocytosis inhibitor), Fucoidan (scavenger receptor inhibitor), Filipin complex (inhibitor of micropinocytosis) and Nocadozole (inhibitor of tubulin depolymerization).Results:As we did observe before, the percentage of Aß positive astrocytes was significantly higher when treated with oligomers compared to fibrils (66% vs 22% P<0.01). The same pattern was also seen for the microglia (79% vs 44% P<0.01). Interestingly, uptake of fibrillar Aß was higher in microglia compared to astrocytes (P1⁄4 0.01), but this was not found for oligomeric Aß uptake. Furthermore, results show that Cytochalasin B was found to inhibit Aß fibril uptake (90% reduction, P<0.01) and to a lesser extent oligomer (50% reduction, P<0.05) uptake in microglia, whereas no effect was seen on Aß uptake by the astrocytes. The other inhibitors of various uptake pathways are currently being tested, and the results will be presented and discussed. Conclusions: These data indicate that Aß can be taken up by both astrocytes and microglia although the cell types seem to use different uptake mechanisms. Aß uptake by microglia probably occurs through endocytosis, whereas astrocytes seem to use another route of internalization.

Impairment of the proteasome on frontal cortex in lewy body dementia

Background: Dementia with Lewy bodies (DLB) and Parkinson’s disease dementia (PDD) account for 10-20% of dementias. The classical neuropathological and neurochemical characteristics include the widespread presence of a-synuclein aggregates in forms of cortical Lewy bodies (LBs). The number of LBs in cortical regions correlates with cognitive impairment in DLB and PDD. Since the ubiquitine-proteasome system (UPS) is the major non-lysosomal pathway for a-synuclein degradation, dysfunction of this system could play a role in the development of a-synuclein aggregates. Methods: The current study examine, mRNA expression (qRT-PCR) and protein levels (western blotting) of a3, ß2, ß6 and Rpt6 subunits of the proteasome and the three associated catalytic activities in post-mortem frontal cortex area (BA 9) from DLB and PDD patients in comparison to age-matched controls. Clinical and pathological data were available for the cases studied (duration of dementia and Parkinsonism, MMSE score, CERAD plaque, Braak stage and a-synuclein scores). Results: Protein expression of the a3 and Rpt6 ATPase sub-unit proteins were significantly lower in only PDD group compared with controls (-40% and -50% respectively), although no differences in the mRNA expression of any of the subunits have been shown between DLB, PDD and control cases. Interestingly all proteasome activities were significantly reduced in PDD patients BA 9 extracts compared to both controls and DLD patients (-58% for chymotrypsin-like, -48% for trypsin-like and -71% for PGPH activities, compared to controls level). In the combined DLB/PDD group, all these three activities and the protein level of Rpt6 subunit correlated inversely with the duration of Parkinsonism. Furthermore the level of Rpt6 expression negatively correlated with the a-synuclein burden in the frontal cortex of these PDD cases (Rs -0.64, p 1⁄4 0.004), and the CERAD plaque score was positively associated with chymotrypsin-like activity (Rs 0.797, < 0.001). Conclusions: These findings indicate that dysfunction of the UPS in the cerebral cortex is a feature of PDD (but not DLB) that occurs at a later stage of the disease process, possibly as a consequence of a-synuclein accumulation. Proteasome activation may therefore have limited utility for the early treatment of Lewy body dementias, however this approach may have benefits in slowing disease progression.