Ana Tereza Di Lorenzo Alho

Brainstem pathology and non-motor symptoms in PD.

Parkinsons disease (PD) is considered a multisystem disorder involving dopaminergic, noradrenergic, serotoninergic, and cholinergic systems, characterized by motor and non-motor symptoms. The causes of the non-motor symptoms in PD are multifactorial and unlikely to be explained by single lesions. However, several evidence link them to damage of specific brainstem nuclei. Numerous brainstem nuclei are engaged in fundamental homeostatic mechanisms, including gastrointestinal regulation, pain perception, mood control, and sleep-wake cycles. In addition, these nuclei are locally interconnected in a complex manner and are subject to supraspinal control. The objective of this review is to provide a better overview of the current knowledge about the consequences of the involvement of specific brainstem nuclei to the most prevalent non-motor symptoms occurring in PD. The multidisciplinary efforts of research directed to these non-nigral brainstem nuclei, in addition to the topographical and chronological spread of the disease - especially in the prodromal stages of PD, are discussed.

Locus coeruleus volume and cell population changes during Alzheimer's disease progression: A stereological study in human postmortem brains with potential implication for early-stage biomarker discovery.

Alzheimers disease (AD) progression follows a specific spreading pattern, emphasizing the need to characterize those brain areas that degenerate first. The brainstems locus coeruleus (LC) is the first area to develop neurofibrillary changes (neurofibrillary tangles [NFTs]).

Sexual Dimorphism in the Human Olfactory Bulb: Females Have More Neurons and Glial Cells than Males

Sex differences in the human olfactory function reportedly exist for olfactory sensitivity, odorant identification and memory, and tasks in which odors are rated based on psychological features such as familiarity, intensity, pleasantness, and others. Which might be the neural bases for these behavioral differences? The number of cells in olfactory regions, and especially the number of neurons, may represent a more accurate indicator of the neural machinery than volume or weight, but besides gross volume measures of the human olfactory bulb, no systematic study of sex differences in the absolute number of cells has yet been undertaken. In this work, we investigate a possible sexual dimorphism in the olfactory bulb, by quantifying postmortem material from 7 men and 11 women (ages 55–94 years) with the isotropic fractionator, an unbiased and accurate method to estimate absolute cell numbers in brain regions. Female bulbs weighed 0.132 g in average, while male bulbs weighed 0.137 g, a non-significant difference; however, the total number of cells was 16.2 million in females, and 9.2 million in males, a significant difference of 43.2%. The number of neurons in females reached 6.9 million, being no more than 3.5 million in males, a difference of 49.3%. The number of non-neuronal cells also proved higher in women than in men: 9.3 million and 5.7 million, respectively, a significant difference of 38.7%. The same differences remained when corrected for mass. Results demonstrate a sex-related difference in the absolute number of total, neuronal and non-neuronal cells, favoring women by 40–50%. It is conceivable that these differences in quantitative cellularity may have functional impact, albeit difficult to infer how exactly this would be, without knowing the specific circuits cells make. However, the reported advantage of women as compared to men may stimulate future work on sex dimorphism of synaptic microcircuitry in the olfactory bulb.

Prevalence of dementia subtypes in a developing country: a clinicopathological study

OBJECTIVES: To assess the distribution of dementia subtypes in Brazil using a population-based clinicopathological study. METHOD: Brains from deceased individuals aged ≥50 years old were collected after the next of kin signed an informed consent form and provided information through standardized questionnaires. Post-mortem clinical diagnoses were established in consensus meetings, and only cases with moderate or severe dementia or without cognitive impairment were included in the analysis. Immunohistochemical neuropathological examinations were performed following the universally accepted guidelines. A diagnosis of Alzheimers disease was made when there were at least both a moderate density of neuritic plaques (Consortium to Establish a Register for Alzheimers disease B or C) and Braak stage III for neurofibrillary tangle distribution. For the diagnosis of vascular dementia, at least three zones or strategic areas had to be affected by infarcts, lacunae, or microinfarcts. RESULTS: From 1,291 subjects, 113 cases were classified as having moderate or severe dementia, and 972 cases were free of cognitive impairment. The neuropathological diagnoses of the dementia sub-group were Alzheimers disease (35.4%), vascular dementia (21.2%), Alzheimers disease plus vascular dementia (13.3%), and other causes of dementia (30.1%). Small-vessel disease, which alone was not considered sufficient for a vascular dementia diagnosis, was present in 38.9% of all of the dementia cases and in 16.8% of the group without cognitive impairment (odds ratio = 2.91; 95% confidence interval, 1.53-5.51), adjusted for age, sex, and education. CONCLUSIONS: The relatively high frequencies of vascular dementia and small-vessel disease in the dementia sub-group constitute relevant findings for public health initiatives because control of vascular risk factors could decrease the prevalence of dementia in developing countries.

Quantifying the accretion of hyperphosphorylated tau in the locus coeruleus and dorsal raphe nucleus: the pathological building blocks of early Alzheimer's Disease

Hyperphosphorylated tau neuronal cytoplasmic inclusions (ht‐NCI) are the best protein correlate of clinical decline in Alzheimers disease (AD). Qualitative evidence identifies ht‐NCI accumulating in the isodendritic core before the entorhinal cortex. Here, we used unbiased stereology to quantify ht‐NCI burden in the locus coeruleus (LC) and dorsal raphe nucleus (DRN), aiming to characterize the impact of AD pathology in these nuclei with a focus on early stages.

A novel approach for integrative studies on neurodegenerative diseases in human brains

Despite a massive research effort to elucidate Alzheimers disease (AD) in recent decades, effective treatment remains elusive. This failure may relate to an oversimplification of the pathogenic processes underlying AD and also lack of understanding of AD progression during its long latent stages. Although evidence shows that the two specific neuropathological hallmarks in AD (neuronal loss and protein accumulation), which are opposite in nature, do not progress in parallel, the great majority of studies have focused on only one of these aspects. Furthermore, research focusing on single structures is likely to render an incomplete picture of AD pathogenesis because as AD involves complete brain networks, potential compensatory mechanisms within the network may ameliorate impairment of the system to a certain extent. Here, we describe an approach for enabling integrative analysis of the dual-nature lesions, simultaneously, in all components of one of the brain networks most vulnerable to AD. This approach is based on significant development of methods previously described mainly by our group that were optimized and complemented for this study. It combines unbiased stereology with immunohistochemistry and immunofluorescence, making use of advanced graphics computing for three-dimensional (3D) volume reconstructions. Although this study was performed in human brainstem and focused in AD, it may be applied to the study of any neurological disease characterized by dual-nature lesions, in humans and animal models. This approach does not require a high level of investment in new equipment and a significant number of specimens can be processed and analyzed within a funding cycle.

Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis

Brain function in normal aging and neurological diseases has long been a subject of interest. With current technology, it is possible to go beyond descriptive analyses to characterize brain cell populations at the molecular level. However, the brain comprises over 100 billion highly specialized cells, and it is a challenge to discriminate different cell groups for analyses. Isolating intact neurons is not feasible with traditional methods, such as tissue homogenization techniques. The advent of laser microdissection techniques promises to overcome previous limitations in the isolation of specific cells. Here, we provide a detailed protocol for isolating and analyzing neurons from postmortem human brain tissue samples. We describe a workflow for successfully freezing, sectioning and staining tissue for laser microdissection. This protocol was validated by mass spectrometric analysis. Isolated neurons can also be employed for western blotting or PCR. This protocol will enable further examinations of brain cell-specific molecular pathways and aid in elucidating distinct brain functions.

Higher Prevalence of TDP-43 Proteinopathy in Cognitively Normal Asians: A Clinicopathological Study on a Multiethnic Sample

Transactive response DNA binding protein 43 (TDP‐43) proteinopathy is the major hallmark of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. It is also present in a subset of Alzheimers disease cases. Recently, few reports showed TDP‐43 changes in cognitively normal elderly. In Caucasians, TDP‐43 proteinopathy independently correlate with cognitive decline. However, it is challenging to establish direct links between cognitive and/or neuropsychiatric symptoms and protein inclusions in neurodegenerative diseases because individual cognitive reserves modify the threshold for clinical disease expression. Cognitive reserve is influenced by demographic, environmental and genetic factors. We investigated the relationships between demographic, clinical and neuropathological variables and TDP‐43 proteinopathy in a large multiethnic sample of cognitively normal elderly. TDP‐43 proteinopathy was identified in 10.5%, independently associated with older age (P = 0.03) and Asian ethnicity (P = 0.002). Asians showed a higher prevalence of TDP‐43 proteinopathy than Caucasians, even after adjustment for sex, age, Braak stage and schooling (odds ratio = 3.50, confidence interval 1.41–8.69, P = 0.007). These findings suggested that Asian older adults may be protected from the clinical manifestation of brain TDP‐43 proteinopathy. Future studies are needed to identify possible race‐related protective factors against clinical expression of TDP‐43 proteinopathies.

Automatic isotropic fractionation for large-scale quantitative cell analysis of nervous tissue

Isotropic fractionation is a quantitative technique that allows reliable estimates of absolute numbers of neuronal and non-neuronal brain cells. However, being fast for single small brains, it requires a long time for processing large brains or many small ones, if done manually. To solve this problem, we developed a machine to automate the method, and tested its efficiency, consistency, and reliability as compared with manual processing. The machine consists of a set of electronically controlled rotation and translation motors coupled to tissue grinders, which automatically transform fixed tissue into homogeneous nuclei suspensions. Speed and torque of the motors can be independently regulated by electronic circuits, according to the volume of tissue being processed and its mechanical resistance to fractionation. To test the machine, twelve paraformaldehyde-fixed rat brains and eight human cerebella were separated into two groups, respectively: one processed automatically and the other, manually. Both pairs of groups (rat and human tissue) followed the same, published protocol of the method. We compared the groups according to nuclei morphology, degree of clustering and number of cells. The machine proved superior for yielding faster results due to simultaneous processing in multiple grinders. Quantitative analysis of machine-processed tissue resulted in similar average numbers of total brain cells, neurons, and non-neuronal cells, statistically similar to the manually processed tissue and equivalent to previously published data. We concluded that the machine is more efficient because it utilizes many homogenizers simultaneously, equally consistent in producing high quality material for counting, and quantitatively reliable as compared to manual processing.

Vascular dementia, an avoidable disease, may be much more common than presumed: A postmortem study in a large population-based series

and 24 dementia with Lewy bodies (DLB). Clinical data comprised MMSE, age at death, duration of disease; neuropathology included immunohistochemistry with assessment of Lewy body and neuritic Braak stages, cortical amyloid plaque load, cerebral amyloid angiopathy, and cerebrovascular lesions (4 degrees). Results: PDD patients were significantly older than PDND (p < 0.01); DLB were slightly younger than PDD; MMSE was PDD < DLB < PDND; duration of illness DLB < PDD < PND (not significant). LB Braak scores were highest in DLB, similar in PDD and PND (5.4/4.5/4.0); neuritic Braak scores PDD < DLB < PDND (4.2/3.8/2.4). Cortical Amyloid plaque load was DLB < PDD < PDND (3.0/2.7/1.0); CAA PDD < DLB < PDND (1.6/1.3/0.2). Striatal Amyloid load in DLB, associated with neuritic Braak stages, was significantly more severe and extensive (70%) than in both PDD (20%) and PDND (only mild in 7.2%). Globus pallidus was negative. Alpha-synuclein deposits in striatum were seen in 76% DLB, 20% PDD and 10% PDND; frequency of striatal tau pathology was DLB < PDD < PDND (70/82/100% negative). Other findings: more severe LB burden in hippocampal CA2/3 area in DLB than in PDD (79 vs 26%). Significant association between cerebrovascular lesions and neuritic Braak stage was seen in PDD but not in DLB and PDND. Conclusions: These data and new amyloid imanging data support a morphologic distinction between DLB and PDD and between PDD and PDND, indicating an influence of AD-related lesions on the progression of the neurodegenerative process and, in particular, on cognitive decline in LBDs. Both these factors appear largely independent from coexisting vascular pathology, except in cases with severe cerebrovascular lesions or those related to neuritic AD pathology. Although synergistic reactions between amyloid, alpha-synuclein and tau protein have been demonstrated by both human pathology, animal models and in vitro studies, the molecular background and the pathophysiologic/clinical impact of these pathologies in LBD await further elucidation.