Oral biopsies in a Portuguese population: A 20-year clinicopathological study in a university clinic.

Background: Performing a biopsy is very important in oral medicine and the anatomopathological examination is fundamental to obtain or to confirm the diagnosis in oral and maxillofacial pathology. The purpose of this study is to analyse the frequency and characteristic patterns of biopsied oromaxillofacial lesions in a Portuguese population. Material and Methods: A descriptive statistical analysis of the data from the anatomopathological reports of the biopsies performed between 1999 and 2019 at the university clinic of the Faculty of Dental Medicine of the University of Lisbon was performed, regarding the patient's gender and age, type of biopsy, location of lesions, clinical and histological diagnosis, and the results were obtained. Association relationships were studied using the chi-square test and the Kruskal-Wallis test to correlate variables. P<0.05 was considered statistically significant. Results: From a total sample of 1448 patients, 826 (57.1%) were female, 610 (42.1%) were male, and 12 (0.8%) had no gender information, with a mean age of 50.14 years (standard deviation ± 17.61). The preferred location was the buccal mucosa, vestibule fundus and alveolar mucosa (20.7%). Benign lesions (BL) were the most common, in 82,8% of the cases, followed by oral potentially malignant disorders (OPMD) in 15,5%, and finally, malignant lesions (ML) in 1.7%. Focal fibrous hyperplasia was the most frequent diagnosis in the total sample (25.6%). In the young group, the most common entity was mucocele (34.0%), with a predominance of the lower lip (32.9%). In OPMD, leukoplakia was the most frequently diagnosed (48,7%). The most common ML was squamous cell carcinoma (92.0%), appearing mainly in the tongue (34.8%). A statistically significant relation between ML and older age was found. Conclusions: This study included biopsies analysed over a period of 20 years, being BL the main pathology to affect the oral cavity. Although less frequent, OPMD and ML should not be neglected and must be correctly diagnosed and treated. Key words:Oral biopsies, Oral and maxillofacial pathology, Oral medicine, Clinicopathological analysis, Epidemiological study, University clinic.

Myosin 1b promotes the formation of post-Golgi carriers by regulating actin assembly and membrane remodelling at the trans-Golgi network.

The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, the cytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have been implicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute to the membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1b regulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, together with the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1 regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation by regulating actin assembly and remodelling TGN membranes.

Myosin 1 controls membrane shape by coupling F-Actin to membrane.

Cellular functions are intimately associated with rapid changes in membrane shape. Different mechanisms interfering with the lipid bilayer, such as the insertion of proteins with amphipatic helices or the association of a protein scaffold, trigger membrane bending. By exerting force on membranes, molecular motors can also contribute to membrane remodeling. Previous studies have shown that actin and myosin 1 participate in the invagination of the plasma membrane during endocytosis while kinesins and dynein with microtubules provide the force to elongate membrane buds at recycling endosomes and at the trans-Golgi network (TGN). Using live cell imaging we have recently shown that a myosin 1 (myosin 1b) regulates the actin dependent post-Golgi traffic of cargo and generates force that controls the assembly of F-actin foci and promotes with the actin cytoskeleton the formation of tubules at the TGN. Our data provide evidence that actin and myosin 1 can regulate membrane remodeling of organelles as well as having an unexpected role in the spatial organization of the actin cytoskeleton. Here, we discuss our results together with the role of actin and other myosins that have been implicated in the traffic of cargo.

Internalized antibodies to the Abeta domain of APP reduce neuronal Abeta and protect against synaptic alterations.

Immunotherapy against beta-amyloid peptide (Abeta) is a leading therapeutic direction for Alzheimer disease (AD). Experimental studies in transgenic mouse models of AD have demonstrated that Abeta immunization reduces Abeta plaque pathology and improves cognitive function. However, the biological mechanisms by which Abeta antibodies reduce amyloid accumulation in the brain remain unclear. We provide evidence that treatment of AD mutant neuroblastoma cells or primary neurons with Abeta antibodies decreases levels of intracellular Abeta. Antibody-mediated reduction in cellular Abeta appears to require that the antibody binds to the extracellular Abeta domain of the amyloid precursor protein (APP) and be internalized. In addition, treatment with Abeta antibodies protects against synaptic alterations that occur in APP mutant neurons.

The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase.

Mutations within the amyloid-beta (Abeta) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer's disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Abeta protofibrils in vitro and intraneuronal Abeta aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased beta-secretase cleavage, as demonstrated by altered levels of N- and C-terminal APP fragments. Although the Arctic mutation is located close to the alpha-secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease-10, a major alpha-secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for alpha-secretase cleavage. As a result, the extent and subcellular location of Abeta formation is changed, as revealed by increased Abeta levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra-Abeta mutations, could relate to altered APP processing with increased steady-state levels of Arctic Abeta, particularly at intracellular locations.

Beta-amyloid accumulation impairs multivesicular body sorting by inhibiting the ubiquitin-proteasome system.

Increasing evidence links intraneuronal beta-amyloid (Abeta42) accumulation with the pathogenesis of Alzheimer's disease (AD). In Abeta precursor protein (APP) mutant transgenic mice and in human AD brain, progressive intraneuronal accumulation of Abeta42 occurs especially in multivesicular bodies (MVBs). We hypothesized that this impairs the MVB sorting pathway. We used the trafficking of the epidermal growth factor receptor (EGFR) and TrkB receptor to investigate the MVB sorting pathway in cultured neurons. We report that, during EGF stimulation, APP mutant neurons demonstrated impaired inactivation, degradation, and ubiquitination of EGFR. EGFR degradation is dependent on translocation from MVB outer to inner membranes, which is regulated by the ubiquitin-proteasome system (UPS). We provide evidence that Abeta accumulation in APP mutant neurons inhibits the activities of the proteasome and deubiquitinating enzymes. These data suggest a mechanism whereby Abeta accumulation in neurons impairs the MVB sorting pathway via the UPS in AD.

Beta-amyloid accumulation in APP mutant neurons reduces PSD-95 and GluR1 in synapses.

Synaptic dysfunction is increasingly viewed as an early manifestation of Alzheimer's disease (AD), but the cellular mechanism by which beta-amyloid (Abeta) may affect synapses remains unclear. Since cultured neurons derived from APP mutant transgenic mice secrete elevated levels of Abeta and parallel the subcellular Abeta accumulation seen in vivo, we asked whether alterations in synapses occur in this setting. We report that cultured Tg2576 APP mutant neurons have selective alterations in pre- and post-synaptic compartments compared to wild-type neurons. Post-synaptic compartments appear fewer in number and smaller, while active pre-synaptic compartments appear fewer in number and enlarged. Among the earliest changes in synaptic composition in APP mutant neurons were reductions in PSD-95, a protein involved in recruiting and anchoring glutamate receptor subunits to the post-synaptic density. In agreement, we observed early reductions in surface expression of glutamate receptor subunit GluR1 in APP mutant neurons. We provide evidence that Abeta is specifically involved in these alterations in synaptic biology, since alterations in PSD-95 and GluR1 are blocked by gamma-secretase inhibition, and since exogenous addition of synthetic Abeta to wild-type neurons parallels changes in synaptic PSD-95 and GluR1 observed in APP mutant neurons.

Regulation of NMDA receptor trafficking by amyloid-beta.

Amyloid-beta peptide is elevated in the brains of patients with Alzheimer disease and is believed to be causative in the disease process. Amyloid-beta reduces glutamatergic transmission and inhibits synaptic plasticity, although the underlying mechanisms are unknown. We found that application of amyloid-beta promoted endocytosis of NMDA receptors in cortical neurons. In addition, neurons from a genetic mouse model of Alzheimer disease expressed reduced amounts of surface NMDA receptors. Reducing amyloid-beta by treating neurons with a gamma-secretase inhibitor restored surface expression of NMDA receptors. Consistent with these data, amyloid-beta application produced a rapid and persistent depression of NMDA-evoked currents in cortical neurons. Amyloid-beta-dependent endocytosis of NMDA receptors required the alpha-7 nicotinic receptor, protein phosphatase 2B (PP2B) and the tyrosine phosphatase STEP. Dephosphorylation of the NMDA receptor subunit NR2B at Tyr1472 correlated with receptor endocytosis. These data indicate a new mechanism by which amyloid-beta can cause synaptic dysfunction and contribute to Alzheimer disease pathology.

Intraneuronal Abeta accumulation and origin of plaques in Alzheimer's disease.

Plaques are a defining neuropathological hallmark of Alzheimer's disease (AD) and the major constituent of plaques, the beta-amyloid peptide (Abeta), is considered to play an important role in the pathophysiology of AD. But the biological origin of Abeta plaques and the mechanism whereby Abeta is involved in pathogenesis have been unknown. Abeta plaques were thought to form from the gradual accumulation and aggregation of secreted Abeta in the extracellular space. More recently, the accumulation of Abeta has been demonstrated to occur within neurons with AD pathogenesis. Moreover, intraneuronal Abeta accumulation has been reported to be critical in the synaptic dysfunction, cognitive dysfunction and the formation of plaques in AD. Here we provide a historical overview on the origin of plaques and a discussion on potential biological and therapeutic implications of intraneuronal Abeta accumulation for AD.

Amyloid-beta oligomers are inefficiently measured by enzyme-linked immunosorbent assay.

Amyloid-beta (Abeta) peptide levels are widely measured by enzyme-linked immunosorbent assay (ELISA) in Alzheimer's disease research. Here, we show that oligomerization of Abeta results in underestimated Abeta ELISA levels. The implications are that comprehensive analysis of soluble Abeta requires either sample pretreatment at denaturing conditions or novel conformation-dependent immunoassays. Our findings might be of relevance for many neurodegenerative disorders in which soluble protein aggregates are the main neurotoxic species.

Increased plaque burden in brains of APP mutant MnSOD heterozygous knockout mice.

A growing body of evidence suggests a relationship between oxidative stress and beta-amyloid (Abeta) peptide accumulation, a hallmark in the pathogenesis of Alzheimer's disease (AD). However, a direct causal relationship between oxidative stress and Abeta pathology has not been established in vivo. Therefore, we crossed mice with a knockout of one allele of manganese superoxide dismutase (MnSOD), a critical antioxidant enzyme, with Tg19959 mice, which overexpress a doubly mutated human beta-amyloid precursor protein (APP). Partial deficiency of MnSOD, which is well established to cause elevated oxidative stress, significantly increased brain Abeta levels and Abeta plaque burden in Tg19959 mice. These results indicate that oxidative stress can promote the pathogenesis of AD and further support the feasibility of antioxidant approaches for AD therapy.

Oligomerization of Alzheimer's beta-amyloid within processes and synapses of cultured neurons and brain.

Multiple lines of evidence implicate beta-amyloid (Abeta) in the pathogenesis of Alzheimer's disease (AD), but the mechanisms whereby Abeta is involved remain unclear. Addition of Abeta to the extracellular space can be neurotoxic. Intraneuronal Abeta42 accumulation is also associated with neurodegeneration. We reported previously that in Tg2576 amyloid precursor protein mutant transgenic mice, brain Abeta42 localized by immunoelectron microscopy to, and accumulated with aging in, the outer membranes of multivesicular bodies, especially in neuronal processes and synaptic compartments. We now demonstrate that primary neurons from Tg2576 mice recapitulate the in vivo localization and accumulation of Abeta42 with time in culture. Furthermore, we demonstrate that Abeta42 aggregates into oligomers within endosomal vesicles and along microtubules of neuronal processes, both in Tg2576 neurons with time in culture and in Tg2576 and human AD brain. These Abeta42 oligomer accumulations are associated with pathological alterations within processes and synaptic compartments in Tg2576 mouse and human AD brains.

Adenosine promotes neuronal recovery from reactive oxygen species induced lesion in rat hippocampal slices.

Reactive oxygen species (ROS) are believed to be involved in the pathogenesis of several neurological disorders. We now tested whether the endogenous neuroprotective substance, adenosine, attenuates the cell damage induced by ROS. In rat hippocampal slices, the xanthine oxidase (40 mU/ml) plus xanthine (1 mM) (X/XO) system produced a 27.8+/-7.3% (n=3) increase in ROS, measured by fluorimetry with 2',7'-dichlorodihydrofluorescein, a 246.9+/-18.4% (n=6) increase in the release of tritiated adenosine, and a decrease in synaptic transmission that fully recovered after washout. In the presence of the adenosine A(1) receptor selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (100 nM), X/XO induced a similar inhibition, however synaptic transmission only recovered to 70.7+/-5.8% of control (n=5). The blockade of A(2A) receptors was devoid of effect (n=4). Adenosine is released by ROS-generating systems, and attenuates the deleterious cellular consequences of ROS through A(1) receptor activation.