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1.
Mol Ther ; 31(2): 409-419, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36369741

RESUMO

The accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) in the brain has been implicated in synapse failure and memory impairment in Alzheimer's disease. Here, we initially show that treatment with NUsc1, a single-chain variable-fragment antibody (scFv) that selectively targets a subpopulation of AßOs and shows minimal reactivity to Aß monomers and fibrils, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by AßOs in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices and inhibited AßO binding to neurons and AßO-induced loss of dendritic spines in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by AßOs and, remarkably, reversed memory deficits in aged APPswe/PS1ΔE9 Alzheimer's disease model mice. These results support the feasibility of immunotherapy using viral vector-mediated gene delivery of NUsc1 or other AßO-specific single-chain antibodies as a potential therapeutic approach in Alzheimer's disease.


Assuntos
Doença de Alzheimer , Anticorpos de Cadeia Única , Camundongos , Ratos , Humanos , Animais , Idoso , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Doença de Alzheimer/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Sinapses/metabolismo , Neurônios/metabolismo , Transtornos da Memória/genética , Transtornos da Memória/terapia
2.
Alzheimers Dement ; 20(2): 995-1012, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37846816

RESUMO

INTRODUCTION: About two-thirds of Alzheimer's Disease (AD) patients are women, who exhibit more severe pathology and cognitive decline than men. Whether biological sex causally modulates the relationship between cholinergic signaling and amyloid pathology remains unknown. METHODS: We quantified amyloid beta (Aß) in male and female App-mutant mice with either decreased or increased cholinergic tone and examined the impact of ovariectomy and estradiol replacement in this relationship. We also investigated longitudinal changes in basal forebrain (cholinergic function) and Aß in elderly individuals. RESULTS: We show a causal relationship between cholinergic tone and amyloid pathology in males and ovariectomized female mice, which is decoupled in ovary-intact and ovariectomized females receiving estradiol. In elderly humans, cholinergic loss exacerbates Aß. DISCUSSION: Our findings emphasize the importance of reflecting human menopause in mouse models. They also support a role for therapies targeting estradiol and cholinergic signaling to reduce Aß. HIGHLIGHTS: Cholinergic tone regulates amyloid beta (Aß) pathology in males and ovariectomized female mice. Estradiol uncouples the relationship between cholinergic tone and Aß. In elderly humans, cholinergic loss correlates with increased Aß in both sexes.


Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Camundongos , Humanos , Feminino , Masculino , Animais , Idoso , Peptídeos beta-Amiloides , Doença de Alzheimer/patologia , Estradiol , Colinérgicos , Precursor de Proteína beta-Amiloide , Camundongos Transgênicos , Modelos Animais de Doenças
3.
J Neurochem ; 2023 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-37804203

RESUMO

Significant evidence suggests that misfolded alpha-synuclein (aSyn), a major component of Lewy bodies, propagates in a prion-like manner contributing to disease progression in Parkinson's disease (PD) and other synucleinopathies. In fact, timed inoculation of M83 hemizygous mice with recombinant human aSyn preformed fibrils (PFF) has shown symptomatic deficits after substantial spreading of pathogenic alpha-synuclein, as detected by markers for the phosphorylation of S129 of aSyn. However, whether accumulated toxicity impact human-relevant cognitive and structural neuroanatomical measures is not fully understood. Here we performed a single unilateral striatal PFF injection in M83 hemizygous mice, and using two assays with translational potential, ex vivo magnetic resonance imaging (MRI) and touchscreen testing, we examined the combined neuroanatomical and behavioral impact of aSyn propagation. In PFF-injected mice, we observed widespread atrophy in bilateral regions that project to or receive input from the injection site using MRI. We also identified early deficits in reversal learning prior to the emergence of motor symptoms. Our findings highlight a network of regions with related cellular correlates of pathology that follow the progression of aSyn spreading, and that affect brain areas relevant for reversal learning. Our experiments suggest that M83 hemizygous mice injected with human PFF provides a model to understand how misfolded aSyn affects human-relevant pre-clinical measures and suggest that these pre-clinical biomarkers could be used to detect early toxicity of aSyn and provide better translational measures between mice and human disease.

4.
Nat Methods ; 17(11): 1139-1146, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32989318

RESUMO

The ability to directly measure acetylcholine (ACh) release is an essential step toward understanding its physiological function. Here we optimized the GRABACh (GPCR-activation-based ACh) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the subsecond response kinetics, physiologically relevant affinity and precise molecular specificity for ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice performing a variety of behaviors.


Assuntos
Acetilcolina/metabolismo , Técnicas Biossensoriais/métodos , Encéfalo/metabolismo , Animais , Animais Geneticamente Modificados , Comportamento Animal/fisiologia , Colinérgicos/farmacologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Corpos Pedunculados/metabolismo , Neurônios/metabolismo , Córtex Olfatório/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Córtex Somatossensorial/metabolismo
5.
FASEB J ; 36(2): e22135, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35032355

RESUMO

In the striatum, cholinergic interneurons (CINs) have the ability to release both acetylcholine and glutamate, due to the expression of the vesicular acetylcholine transporter (VAChT) and the vesicular glutamate transporter 3 (VGLUT3). However, the relationship these neurotransmitters have in the regulation of behavior is not fully understood. Here we used reward-based touchscreen tests in mice to assess the individual and combined contributions of acetylcholine/glutamate co-transmission in behavior. We found that reduced levels of the VAChT from CINs negatively impacted dopamine signalling in response to reward, and disrupted complex responses in a sequential chain of events. In contrast, diminished VGLUT3 levels had somewhat opposite effects. When mutant mice were treated with haloperidol in a cue-based task, the drug did not affect the performance of VAChT mutant mice, whereas VGLUT3 mutant mice were highly sensitive to haloperidol. In mice where both vesicular transporters were deleted from CINs, we observed altered reward-evoked dopaminergic signalling and behavioral deficits that resemble, but were worse, than those in mice with specific loss of VAChT alone. These results demonstrate that the ability to secrete two different neurotransmitters allows CINs to exert complex modulation of a wide range of behaviors.


Assuntos
Acetilcolina/metabolismo , Colinérgicos/metabolismo , Corpo Estriado/metabolismo , Ácido Glutâmico/metabolismo , Interneurônios/metabolismo , Animais , Dopamina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurotransmissores/metabolismo , Transdução de Sinais/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo
6.
Am J Physiol Cell Physiol ; 322(4): C794-C801, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35264016

RESUMO

It is well known that cholinergic hypofunction contributes to cardiac pathology, yet, the mechanisms involved remain unclear. Our previous study has shown that genetically engineered model of cholinergic deficit, the vesicular acetylcholine transporter knockdown homozygous (VAChT KDHOM) mice, exhibit pathological cardiac remodeling and a gradual increase in cardiac mass with aging. Given that an increase in cardiac mass is often caused by adrenergic hyperactivity, we hypothesized that VAChT KDHOM mice might have an increase in cardiac norepinephrine (NE) levels. We thus investigated the temporal changes in NE content in the heart from 3-, 6-, and 12-mo-old VAChT mutants. Interestingly, mice with cholinergic hypofunction showed a gradual elevation in cardiac NE content, which was already increased at 6 mo of age. Consistent with this finding, 6-mo-old VAChT KDHOM mice showed enhanced sympathetic activity and a greater abundance of tyrosine hydroxylase positive sympathetic nerves in the heart. VAChT mutants exhibited an increase in peak calcium transient, and mitochondrial oxidative stress in cardiomyocytes along with enhanced G protein-coupled receptor kinase 5 (GRK5) and nuclear factor of activated T-cells (NFAT) staining in the heart. These are known targets of adrenergic signaling in the cell. Moreover, vagotomized-mice displayed an increase in cardiac NE content confirming the data obtained in VAChT KDHOM mice. Establishing a causal relationship between acetylcholine and NE, VAChT KDHOM mice treated with pyridostigmine, a cholinesterase inhibitor, showed reduced cardiac NE content, rescuing the phenotype. Our findings unveil a yet unrecognized role of cholinergic signaling as a modulator of cardiac NE, providing novel insights into the mechanisms that drive autonomic imbalance.


Assuntos
Colinérgicos , Norepinefrina , Adrenérgicos , Animais , Camundongos , Miócitos Cardíacos , Proteínas Vesiculares de Transporte de Acetilcolina/genética
7.
Hippocampus ; 32(10): 731-751, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36123765

RESUMO

We hypothesize that hippocampal local field potentials in acetylcholine (ACh)-deficient mutant mice, compared to wild-type (WT) mice, will show lower sensitivity to muscarinic cholinergic antagonist scopolamine (5 mg/kg i.p.) but higher sensitivity to NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP, 10 mg/kg i.p.). Recordings were made during walk and awake-immobility (IMM) in WT mice, and in mice with forebrain knockout (KO) of the vesicular acetylcholine transporter (VAChT) gene, or heterozygous knockdown of VAChT gene (KD). Scopolamine or CPP did not significantly alter walk theta frequency, which was higher in KD than WT/KO mice. Scopolamine decreased theta power peak rise during walk in WT/KD mice but not in KO mice, while CPP suppressed theta peak rise more in WT/KO mice than KD mice. During IMM, scopolamine decreased gamma1 (γ1, 30-58 Hz) power more in KD/WT mice than KO mice, while delta (1-4 Hz) power and delta-gamma cross-frequency coherence (CFC) were increased in all mouse groups during IMM or walk. During walk, scopolamine increased delta and beta (13-30 Hz) power and decreased gamma2 (γ2, 62-100 Hz) power and theta-γ2 CFC more in WT/KD than KO mice. Theta-γ2, but not theta-γ1, CFC increased with theta-peak-frequency in WT/KD mice, and was suppressed by scopolamine at high theta (8-10 Hz) frequency; theta-γ2 CFC in KO mice was not significantly altered by scopolamine. CPP decreased beta and gamma power more in KD/KO mice compared to WT mice, while delta power and delta-gamma CFC were increased in all mouse groups. ACh deficiency exacerbates the attenuation of beta and gamma power by CPP. We conclude that both muscarinic and NMDA transmission contribute toward hippocampal theta, beta, and gamma power, and a decrease in gamma power or theta-gamma CFC may be associated with loss of arousal and cognitive functions.


Assuntos
Acetilcolina , Receptores de N-Metil-D-Aspartato , Animais , Hipocampo/fisiologia , Camundongos , Camundongos Knockout , Antagonistas Muscarínicos/farmacologia , N-Metilaspartato , Escopolamina/farmacologia , Proteínas Vesiculares de Transporte de Acetilcolina
8.
Acta Neuropathol ; 144(5): 881-910, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36121476

RESUMO

The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90.


Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas Intrinsicamente Desordenadas , alfa-Sinucleína/metabolismo , Animais , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Chaperonas Moleculares/metabolismo , Fosfoproteínas , Ubiquitinas , alfa-Sinucleína/toxicidade
9.
FASEB J ; 35(5): e21594, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33908654

RESUMO

Protein misfolding is a central feature of most neurodegenerative diseases. Molecular chaperones can modulate the toxicity associated with protein misfolding, but it remains elusive which molecular chaperones and co-chaperones interact with specific misfolded proteins. TDP-43 misfolding and inclusion formation are a hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Using yeast and mammalian neuronal cells we find that Hsp90 and its co-chaperone Sti1 have the capacity to alter TDP-43 misfolding, inclusion formation, aggregation, and cellular toxicity. Our data also demonstrate that impaired Hsp90 function sensitizes cells to TDP-43 toxicity and that Sti1 specifically interacts with and strongly modulates TDP-43 toxicity in a dose-dependent manner. Our study thus uncovers a previously unrecognized tie between Hsp90, Sti1, TDP-43 misfolding, and cellular toxicity.


Assuntos
Apoptose , Proteínas de Ligação a DNA/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/fisiologia , Dobramento de Proteína , Saccharomyces cerevisiae/metabolismo , Proteinopatias TDP-43/patologia , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Proteínas de Choque Térmico HSP90/genética , Células HeLa , Humanos , Corpos de Inclusão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteinopatias TDP-43/etiologia
10.
Eur J Neurosci ; 54(6): 6075-6092, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34308559

RESUMO

Disruptions in social behaviour are prevalent in many neuropsychiatric disorders such as schizophrenia, bipolar disorder and autism spectrum disorders. However, the underlying neurochemical regulation of social behaviour is still not well understood. The central cholinergic system has been proposed to contribute to the regulation of social behaviour. For instance, decreased global levels of acetylcholine release in the brain leads to decreased social interaction and an impairment of social memory in mice. Nonetheless, it has been difficult to ascertain the specific brain areas where cholinergic signalling influences social preference and social memory. In this study, we investigated the impact of different forebrain cholinergic regions on social behaviour by examining mouse lines that differ in their regional expression level of the vesicular acetylcholine transporter-the protein that regulates acetylcholine secretion. We found that when cholinergic signalling is highly disrupted in the striatum, hippocampus, cortex and amygdala mice have intact social preference but are impaired in social memory, as they cannot remember a familiar conspecific nor recognize a novel one. A similar pattern emerges when acetylcholine release is disrupted mainly in the striatum, cortex, and amygdala; however, the ability to recognize novel conspecifics is retained. In contrast, cholinergic signalling of the striatum and amygdala does not appear to significantly contribute to the modulation of social memory and social preference. Furthermore, we demonstrated that increasing global cholinergic tone does not increase social behaviours. Together, these data suggest that cholinergic transmission from the hippocampus and cortex are important for regulating social memory.


Assuntos
Prosencéfalo Basal , Acetilcolina , Animais , Colinérgicos , Hipocampo/metabolismo , Masculino , Camundongos , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo
11.
Anesthesiology ; 134(4): 588-606, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33635947

RESUMO

BACKGROUND: Cholinergic drugs are known to modulate general anesthesia, but anesthesia responses in acetylcholine-deficient mice have not been studied. It was hypothesized that mice with genetic deficiency of forebrain acetylcholine show increased anesthetic sensitivity to isoflurane and ketamine and decreased gamma-frequency brain activity. METHODS: Male adult mice with heterozygous knockdown of vesicular acetylcholine transporter in the brain or homozygous knockout of the transporter in the basal forebrain were compared with wild-type mice. Hippocampal and frontal cortical electrographic activity and righting reflex were studied in response to isoflurane and ketamine doses. RESULTS: The loss-of-righting-reflex dose for isoflurane was lower in knockout (mean ± SD, 0.76 ± 0.08%, n = 18, P = 0.005) but not knockdown (0.78 ± 0.07%, n = 24, P = 0.021), as compared to wild-type mice (0.83 ± 0.07%, n = 23), using a significance criterion of P = 0.017 for three planned comparisons. Loss-of-righting-reflex dose for ketamine was lower in knockout (144 ± 39 mg/kg, n = 14, P = 0.006) but not knockdown (162 ± 32 mg/kg, n = 20, P = 0.602) as compared to wild-type mice (168 ± 24 mg/kg, n = 21). Hippocampal high-gamma (63 to 100 Hz) power after isoflurane was significantly lower in knockout and knockdown mice compared to wild-type mice (isoflurane-dose and mouse-group interaction effect, F[8,56] = 2.87, P = 0.010; n = 5 to 6 mice per group). Hippocampal high-gamma power after ketamine was significantly lower in both knockout and knockdown mice when compared to wild-type mice (interaction effect F[2,13] = 6.06, P = 0.014). The change in frontal cortical gamma power with isoflurane or ketamine was not statistically different among knockout, knockdown, and wild-type mice. CONCLUSIONS: These findings suggest that forebrain cholinergic neurons modulate behavioral sensitivity and hippocampal gamma activity during isoflurane and ketamine anesthesia.


Assuntos
Acetilcolina/farmacologia , Anestésicos Inalatórios/farmacologia , Isoflurano/farmacologia , Ketamina/farmacologia , Prosencéfalo/efeitos dos fármacos , Animais , Masculino , Camundongos , Camundongos Knockout , Modelos Animais
12.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299169

RESUMO

(1) Background: The lung cholinergic pathway is important for controlling pulmonary inflammation in acute lung injury, a condition that is characterized by a sudden onset and intense inflammation. This study investigated changes in the expression levels of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) in the lung during acute lung injury. (2) Methods: acute lung injury (ALI) was induced in wild-type and cholinergic-deficient (VAChT-KDHOM) mice using intratracheal lipopolysaccharide (LPS) instillation with or without concurrent treatment with nicotinic ligands. Bronchoalveolar lavage fluid was collected to evaluate markers of inflammation, and then the lung was removed and processed for isolation of membrane fraction and determination of acetylcholine receptors level using radioligand binding assays. (3) Results: LPS-induced increase in lung inflammatory markers (e.g., neutrophils and IL-1ß) was significantly higher in VAChT-KDHOM than wild-type mice. In contrast, LPS treatment resulted in a significant increase in lung's α7 nicotinic receptor level in wild-type, but not in VAChT-KDHOM mice. However, treatment with PNU 282987, a selective α7 nicotinic receptor agonist, restored VAChT-KDHOM mice's ability to increase α7 nicotinic receptor levels in response to LPS-induced acute lung injury and reduced lung inflammation. LPS also increased muscarinic receptors level in VAChT-KDHOM mice, and PNU 282987 treatment reduced this response. (4) Conclusions: Our data indicate that the anti-inflammatory effects of the lung cholinergic system involve an increase in the level of α7 nicotinic receptors. Pharmacological agents that increase the expression or the function of lung α7 nicotinic receptors have potential clinical uses for treating acute lung injury.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Colinérgicos/metabolismo , Pneumonia/prevenção & controle , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Citocinas/metabolismo , Masculino , Camundongos , Agonistas Nicotínicos/farmacologia , Pneumonia/etiologia , Pneumonia/metabolismo , Pneumonia/patologia , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Receptor Nicotínico de Acetilcolina alfa7/genética
13.
J Neurochem ; 153(6): 727-758, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31562773

RESUMO

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749.


Assuntos
Envelhecimento/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/deficiência , Hipocampo/metabolismo , Chaperonas Moleculares/metabolismo , Adaptação Fisiológica/fisiologia , Envelhecimento/genética , Animais , Células-Tronco Embrionárias/metabolismo , Técnicas de Inativação de Genes/métodos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico/genética , Hipocampo/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/genética , Neurônios/metabolismo
14.
FASEB J ; 33(6): 7018-7036, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30857416

RESUMO

The pedunculopontine tegmental nucleus (PPT) and laterodorsal tegmental nucleus (LDT) are heterogeneous brainstem structures that contain cholinergic, glutamatergic, and GABAergic neurons. PPT/LDT neurons are suggested to modulate both cognitive and noncognitive functions, yet the extent to which acetylcholine (ACh) signaling from the PPT/LDT is necessary for normal behavior remains uncertain. We addressed this issue by using a mouse model in which PPT/LDT cholinergic signaling is highly decreased by selective deletion of the vesicular ACh transporter (VAChT) gene. This approach interferes exclusively with ACh signaling, leaving signaling by other neurotransmitters from PPT/LDT cholinergic neurons intact and sparing other cells. VAChT mutants were examined on different PPT/LDT-associated cognitive domains. Interestingly, VAChT mutants showed no attentional deficits and only minor cognitive flexibility impairments while presenting large deficiencies in both spatial and cued Morris water maze (MWM) tasks. Conversely, working spatial memory determined with the Y-maze and spatial memory measured with the Barnes maze were not affected, suggesting that deficits in MWM were unrelated to spatial memory abnormalities. Supporting this interpretation, VAChT mutants exhibited alterations in anxiety-like behavior and increased corticosterone levels after exposure to the MWM, suggesting altered stress response. Thus, PPT/LDT VAChT-mutant mice present little cognitive impairment per se, yet they exhibit increased susceptibility to stress, which may lead to performance deficits in more stressful conditions.-Janickova, H., Kljakic, O., Rosborough, K., Raulic, S., Matovic, S., Gros, R., Saksida, L. M., Bussey, T. J., Inoue, W., Prado, V. F., Prado, M. A. M. Selective decrease of cholinergic signaling from pedunculopontine and laterodorsal tegmental nuclei has little impact on cognition but markedly increases susceptibility to stress.


Assuntos
Cognição/fisiologia , Núcleos Laterais do Tálamo/fisiologia , Núcleo Tegmental Pedunculopontino/fisiologia , Estresse Fisiológico , Animais , Atenção , Corticosterona/sangue , Regulação da Expressão Gênica , Proteínas Vesiculares de Transporte de Acetilcolina/genética
15.
Brain Behav Immun ; 88: 791-801, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32434046

RESUMO

Microglia express muscarinic G protein-coupled receptors (GPCRs) that sense cholinergic activity and are activated by acetylcholine to potentially regulate microglial functions. Knowledge about how distinct types of muscarinic GPCR signaling regulate microglia function in vivo is still poor, partly due to the fact that some of these receptors are also present in astrocytes and neurons. We generated mice expressing the hM3Dq Designer Receptor Exclusively Activated by Designer Drugs (DREADD) selectively in microglia to investigate the role of muscarinic M3Gq-linked signaling. We show that activation of hM3Dq using clozapine N-oxide (CNO) elevated intracellular calcium levels and increased phagocytosis of FluoSpheres by microglia in vitro. Interestingly, whereas acute treatment with CNO increased synthesis of cytokine mRNA, chronic treatment attenuated LPS-induced cytokine mRNA changes in the brain. No effect of CNO on cytokine expression was observed in DREADD-negative mice. Interestingly, CNO activation of M3Dq in microglia was able to attenuate LPS-mediated decrease in social interactions. These results suggest that chronic activation of M3 muscarinic receptors (the hM3Dq progenitor) in microglia, and potentially other Gq-coupled GPCRs, can trigger an inflammatory-like response that preconditions microglia to decrease their response to further immunological challenges. Our results indicate that hM3Dq can be a useful tool to modulate neuroinflammation and study microglial immunological memory in vivo, which may be applicable for manipulations of neuroinflammation in neurodegenerative and psychiatric diseases.


Assuntos
Clozapina , Microglia , Acetilcolina , Animais , Clozapina/farmacologia , Masculino , Camundongos , Neurônios , Receptores Acoplados a Proteínas G , Transdução de Sinais
16.
Ecotoxicol Environ Saf ; 167: 494-504, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30368143

RESUMO

Endogenous acetylcholine (ACh), which depends of the levels of vesicular ACh transport (VAChT) to be released, is the central mediator of the cholinergic anti-inflammatory system. ACh controls the release of cytokine in different models of inflammation. Diesel exhaust particles (DEP) are one of the major environmental pollutants produced in large quantity by automotive engines in urban center. DEP bind the lung parenchyma and induce inflammation. We evaluated whether cholinergic dysfunction worsens DEP-induced lung inflammation. Male mice with decreased ACh release due to reduced expression of VAChT (VAChT-KD mice) were submitted to DEP exposure for 30 days (3 mg/mL of DEP, once a day, five days a week) or saline. Pulmonary function and inflammation as well as extracellular matrix fiber deposition were evaluated. Additionally, airway and nasal epithelial mucus production were quantified. We found that DEP instillation worsened lung function and increased lung inflammation. Higher levels of mononuclear cells were observed in the peripheral blood of both wild-type (WT) and VAChT-KD mice. Also, both wild-type (WT) and VAChT-KD mice showed an increase in macrophages in bronchoalveolar lavage fluid (BALF) as well as increased expression of IL-4, IL-6, IL-13, TNF-α, and NF-κB in lung cells. The collagen fiber content in alveolar septa was also increased in both genotypes. On the other hand, we observed that granulocytes were increased only in VAChT-KD peripheral blood. Likewise, increased BALF lymphocytes and neutrophils as well as increased elastic fibers in alveolar septa, airway neutral mucus, and nasal epithelia acid mucus were observed only in VAChT-KD mice. The cytokines IL-4 and TNF-α were also higher in VAChT-KD mice compared with WT mice. In conclusion, decreased ability to release ACh exacerbates some of the lung alterations induced by DEP in mice, suggesting that VAChT-KD animals are more vulnerable to the effects of DEP in the lung.


Assuntos
Pulmão/efeitos dos fármacos , Emissões de Veículos/toxicidade , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Animais , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/genética , Citocinas/metabolismo , Pulmão/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Tecido Parenquimatoso/efeitos dos fármacos , Tecido Parenquimatoso/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/diagnóstico , Proteínas Vesiculares de Transporte de Acetilcolina/deficiência , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo
17.
J Neurochem ; 145(1): 68-79, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29265373

RESUMO

Stress-inducible phosphoprotein 1 (STI1) acts as a neuroprotective factor in the ischemic brain and its levels are increased following ischemia. Previous work has suggested that some of these STI1 actions in a stroke model depend on the recruitment of bone marrow-derived stem cells to improve outcomes after ischemic insult. However, STI1 can directly increase neuroprotective signaling in neurons by engaging with the cellular prion protein (PrPC ) and activating α7 nicotinic acetylcholine receptors (α7nAChR). Given that α7nAChR activation has also been involved in neuroprotection in stroke, it is possible that STI1 can have direct actions on neurons to prevent deleterious consequences of ischemic insults. Here, we tested this hypothesis by exposing primary neuronal cultures to 1-h oxygen-glucose deprivation (OGD) and reperfusion and assessing signaling pathways activated by STI1/PrPC . Our results demonstrated that STI1 treatment significantly decreased apoptosis and cell death in mouse neurons submitted to OGD in a manner that was dependent on PrPC and α7nAChR, but also on the activin A receptor 1 (ALK2), which has emerged as a signaling partner of STI1. Interestingly, pharmacological inhibition of the ALK2 receptor prevented neuroprotection by STI1, while activation of ALK2 receptors by bone morphogenetic protein 4 (BMP4) either before or after OGD was effective in decreasing neuronal death induced by ischemia. We conclude that PrPC /STI1 engagement and its subsequent downstream signaling cascades involving α7nAChR as well as the ALK2 receptor may be activated in neurons by increased levels of STI1. This signaling pathway protects neurons from ischemic insults.


Assuntos
Isquemia Encefálica/metabolismo , Proteínas de Choque Térmico/metabolismo , Neuroproteção/fisiologia , Proteínas Priônicas/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Animais , Apoptose/fisiologia , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Transdução de Sinais/fisiologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
18.
FASEB J ; 31(1): 320-332, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27729414

RESUMO

Nicotinic α-7 acetylcholine receptor (nAChRα7) is a critical regulator of cholinergic anti-inflammatory actions in several diseases, including acute respiratory distress syndrome (ARDS). Given the potential importance of α7nAChR as a therapeutic target, we evaluated whether PNU-282987, an α7nAChR agonist, is effective in protecting the lung against inflammation. We performed intratracheal instillation of LPS to generate acute lung injury (ALI) in C57BL/6 mice. PNU-282987 treatment, either before or after ALI induction, reduced neutrophil recruitment and IL-1ß, TNF-α, IL-6, keratinocyte chemoattractant (KC), and IL-10 cytokine levels in the bronchoalveolar lavage fluid (P < 0.05). In addition, lung NF-κB phosphorylation decreased, along with collagen fiber deposition and the number of matrix metalloproteinase-9+ and -2+ cells, whereas the number of tissue inhibitor of metalloproteinase-1+ cells increased (P < 0.05). PNU-282987 treatment also reduced lung mRNA levels and the frequency of M1 macrophages, whereas cells expressing the M2-related markers CD206 and IL-10 increased, suggesting changes in the macrophage profile. Finally, PNU-282987 improved lung function in LPS-treated animals. The collective results suggest that PNU-282987, an agonist of α7nAChR, reduces LPS-induced experimental ALI, thus supporting the notion that drugs that act on α7nAChRs should be explored for ARDS treatment in humans.-Pinheiro, N. M., Santana, F. P. R., Almeida, R. R., Guerreiro, M., Martins, M. A., Caperuto, L. C., Câmara, N. O. S., Wensing, L. A., Prado, V. F., Tibério, I. F. L. C., Prado, M. A. M., Prado, C. M. Acute lung injury is reduced by the α7nAChR agonist PNU-282987 through changes in the macrophage profile.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Benzamidas/uso terapêutico , Compostos Bicíclicos com Pontes/uso terapêutico , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Líquido da Lavagem Broncoalveolar , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Masculino , Camundongos , RNA/genética , RNA/metabolismo
19.
Cereb Cortex ; 27(2): 1615-1628, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-26803167

RESUMO

Cholinergic dysfunction has been associated with cognitive abnormalities in a variety of neurodegenerative and neuropsychiatric diseases. Here we tested how information processing is regulated by cholinergic tone in genetically modified mice targeting the vesicular acetylcholine transporter (VAChT), a protein required for acetylcholine release. We measured long-term potentiation of Schaffer collateral-CA1 synapses in vivo and assessed information processing by using a mouse touchscreen version of paired associates learning task (PAL). Acquisition of information in the mouse PAL task correlated to levels of hippocampal VAChT, suggesting a critical role for cholinergic tone. Accordingly, synaptic plasticity in the hippocampus in vivo was disturbed, but not completely abolished, by decreased hippocampal cholinergic signaling. Disrupted forebrain cholinergic signaling also affected working memory, a result reproduced by selectively decreasing VAChT in the hippocampus. In contrast, spatial memory was relatively preserved, whereas reversal spatial memory was sensitive to decreased hippocampal cholinergic signaling. This work provides a refined roadmap of how synaptically secreted acetylcholine influences distinct behaviors and suggests that distinct forms of cognitive processing may be regulated in different ways by cholinergic activity.


Assuntos
Acetilcolina/metabolismo , Hipocampo/fisiologia , Memória de Curto Prazo/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Animais , Potenciação de Longa Duração/fisiologia , Camundongos Transgênicos , Prosencéfalo/metabolismo , Memória Espacial/fisiologia , Sinapses/metabolismo
20.
Cereb Cortex ; 27(7): 3553-3567, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27312991

RESUMO

The relationship between long-term cholinergic dysfunction and risk of developing dementia is poorly understood. Here we used mice with deletion of the vesicular acetylcholine transporter (VAChT) in the forebrain to model cholinergic abnormalities observed in dementia. Whole-genome RNA sequencing of hippocampal samples revealed that cholinergic failure causes changes in RNA metabolism. Remarkably, key transcripts related to Alzheimer's disease are affected. BACE1, for instance, shows abnormal splicing caused by decreased expression of the splicing regulator hnRNPA2/B1. Resulting BACE1 overexpression leads to increased APP processing and accumulation of soluble Aß1-42. This is accompanied by age-related increases in GSK3 activation, tau hyperphosphorylation, caspase-3 activation, decreased synaptic markers, increased neuronal death, and deteriorating cognition. Pharmacological inhibition of GSK3 hyperactivation reversed deficits in synaptic markers and tau hyperphosphorylation induced by cholinergic dysfunction, indicating a key role for GSK3 in some of these pathological changes. Interestingly, in human brains there was a high correlation between decreased levels of VAChT and hnRNPA2/B1 levels with increased tau hyperphosphorylation. These results suggest that changes in RNA processing caused by cholinergic loss can facilitate Alzheimer's-like pathology in mice, providing a mechanism by which decreased cholinergic tone may increase risk of dementia.


Assuntos
Acetilcolina/metabolismo , Doença de Alzheimer/patologia , Regulação da Expressão Gênica/genética , Hipocampo/metabolismo , RNA/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/deficiência , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Hipocampo/citologia , Humanos , Deficiências da Aprendizagem/etiologia , Deficiências da Aprendizagem/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA/genética , Tiazóis/farmacologia , Fator Nuclear 1 de Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo , Ureia/análogos & derivados , Ureia/farmacologia , Proteínas Vesiculares de Transporte de Acetilcolina/genética
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