Background

The role of the cerebellum in coordinating mental activity is supported by its connections with cerebral regions involved in cognitive/affective functioning, with decreased activities on functional neuroimaging observed in the cerebellum of schizophrenia patients performing mental tasks. Brain-derived neurotrophic factor (BDNF)-induced activation of tyrosine kinase B (TrkB) is essential to synaptic plasticity. We hypothesized that alterations in BDNF and TrkB expression in the cerebellum were associated with schizophrenia and affective disorders.

Methods

We employed immunohistochemistry and immunoblotting to quantify protein expression of BDNF and TrkB in the cerebellum of patients with schizophrenia, bipolar disorder, and major depression compared to controls (n=15 each).

Results

While TrkB immunoreactivity in each of the molecular and granule-cell layers was reduced in all 3 disease groups (12–34%) compared to the control (P=0.018 and 0.038, respectively, ANOVA), only the reduction in bipolar disorder remained statistically significant upon Tukey-Kramer post hoc analyses (P=0.019 and 0.021, respectively). Apparent decreases in BDNF immunoreactivity in all 3 disease groups (12–30%) compared to the control were not statistically significant. TrkB immunoreactivity was not significantly associated with any of the demographic, clinical, and postmortem variables. Immunoblotting displayed an 85-kDa TrkB-immunoreactive band, consistent with a truncated isoform, in all 60 cases.

Limitations

On immunoblotting, apparent decreases in 85-kDa-TrkB levels in all 3 disease groups compared to the control were not statistically significant.

Conclusions

Our finding of reduced TrkB expression in bipolar disorder suggests that dysregulation of TrkB-mediated neurotrophin signaling in the cerebellum may play a role in the pathophysiology of this disease.

Background

The analysis of co-localized protein expression in a tissue section is often conducted with immunofluorescence histochemical staining which is typically visualized in localized regions. On the other hand, chromogenic immunohistochemical staining, in general, is not suitable for the detection of protein co-localization. Here, we developed a new protocol, based on chromogenic immunohistochemical stain, for system-wide detection of protein co-localization and differential expression.

Methodology/Principal Findings

In combination with a removable chromogenic stain, an efficient antibody stripping method was developed to enable sequential immunostaining with different primary antibodies regardless of antibody's host species. Sections were scanned after each staining, and the images were superimposed together for the detection of protein co-localization and differential expression. As a proof of principle, differential expression and co-localization of glutamic acid decarboxylase67 (GAD67) and parvalbumin proteins was examined in mouse cortex.

Conclusions/Significance

All parvalbumin-containing neurons express GAD67 protein, and GAD67-positive neurons that do not express parvalbumin were readily visualized from thousands of other neurons across mouse cortex. The method provided a global view of protein co-localization as well as differential expression across an entire tissue section. Repeated use of the same section could combine assessments of co-localization and differential expression of multiple proteins.

The occurrence and progression of cerebral β-amyloid angiopathy (CAA) and β-amyloid plaques in sporadic Alzheimer's disease may be attributed to aging-related deficiencies in β-amyloid drainage along cerebral perivascular pathways. To elucidate high-definition characteristics of cerebral β-amyloid deposition, we performed immunogold silver staining for β-amyloid-40 and β-amyloid-42 on semithin LR White-embedded tissue sections from 7 Alzheimer's disease/severe CAA, 9 Alzheimer's disease/mild CAA, 5 old control and 4 young control autopsy brains. In vessel walls, β-amyloid-40 and β-amyloid-42 deposits were unevenly distributed along the adventitia and among the medial smooth muscle cells. β-Amyloid-40 immunoreactivity appeared greater than that of β-amyloid-42 in vessel walls, with β-amyloid-42 being preferentially located on their abluminal regions. In capillary walls, either β-amyloid-40 or β-amyloid-42 deposits or both were present in 6 of 7 severe CAA and 1 of 9 mild CAA cases, with a marked variation in thickness and focally abluminal excrescences. In 5 of 7 severe CAA cases, a subset of β-amyloid-laden capillaries revealed either β-amyloid-40 or β-amyloid-42 deposits or both radiating from their walls into the surrounding neuropil (“pericapillary deposits”). No vascular β-amyloid-40 or β-amyloid-42 deposits were observed in any of the controls. In conclusion, the patterns of β-amyloid-42 and β-amyloid-40 immunoreactivity in vessel walls suggest that β-amyloid deposits occur in the vascular basement membranes along cerebral perivascular drainage pathways, extending from cortical capillaries to leptomeningeal arteries. The presence of pericapillary β-amyloid deposits suggests that a subset of β-amyloid plaques originate from β-amyloid-laden capillaries, particularly in Alzheimer's disease brains that exhibit preferential capillary CAA involvement.

Cerebral amyloid angiopathy (CAA) is common in elderly individuals, especially those affected with Alzheimer's disease. To investigate whether the presence of severe CAA (SCAA) in the brains of demented patients was associated with a higher burden of old microinfarcts than those with mild CAA (MCAA), 18 brains with SCAA were compared to 21 brains with MCAA. Immunohistochemistry for CD68 was employed to highlight old microinfarcts in tissue blocks from various brain regions. Old microinfarcts, manually counted by light microscopy, were present in 14 of 18 SCAA brains, and in 7 of 21 MCAA brains (P = 0.01, 2-tailed Fisher’s exact test). The average number of old microinfarcts across geographic regions in each brain ranged from 0 to 1.95 (mean rank 24.94, sum of ranks 449) in the SCAA group, and from 0 to 0.35 (mean rank 15.76, sum of ranks 331) in the MCAA group (P = 0.008, 2-tailed Mann-Whitney U test). Frequent old microinfarcts in demented individuals with severe CAA may contribute a vascular component to the cognitive impairment in these patients.

Constitutive macroautophagy involved in the turnover of defective long-lived proteins and organelles is crucial for neuronal homeostasis. We hypothesized that macroautophagic dysregulation in selective brain regions was associated with memory impairment in aged mice. We used the single-trial object recognition test to measure short-term memory in 18 aged mice compared to 22 young mice and employed immunohistochemistry to assess cellular distribution of proteins involved in the selective degradation of ubiquitinated proteins via macroautophagy. Values of the discrimination ratio (DR, a measure of short-term recognition memory performance) in aged mice were significantly lower than those in young mice (median, 0.54 vs. 0.67; p = 0.005, U test). Almost exclusively in aged mice, there were clusters of puncta immunoreactive for microtubule-associated protein 1 light chain 3 (LC3), ubiquitin- and LC3-binding protein p62, and ubiquitin in neuronal processes predominantly in the hippocampal formation, olfactory bulb/tubercle, and cerebellar cortex. The hippocampal burden of clustered puncta immunoreactive for LC3 and p62 exhibited inverse linear correlations with DR in aged mice (ρ = −0.48 and −0.55, p = 0.044 and 0.018, respectively, Spearman’s rank correlation). These findings suggest that increased accumulation of autophagosomes within neuronal processes in selective brain regions is characteristic of aging. The dysregulation of macroautophagy can adversely affect the turnover of aggregate-prone proteins and defective organelles, which may contribute to memory impairment in aged mice.

Patients infected with human immunodeficiency virus (HIV) have a higher risk of developing major depressive disorder (MDD) than the general population. Immunophilins FKBP51 and FKBP52 are expressed in cortical neurons and regulate the function of the glucocorticoid receptor (GR). Previous reports have shown that genetic variants in the FKBP5 gene encoding FKBP51 are linked to psychiatric disorders. We sought to determine whether immunophilins are upregulated in HIV infection. To determine whether FKBP52 and FKBP51 are associated with MDD and/or HIV, we compared protein and gene expression in autopsy tissues from the frontal cortical gray matter. The study cases were divided into five groups: control, MDD, MDD with psychosis, HIV+, and HIV+ with MDD. Gene expression and protein levels were determined by real-time PCR and Western blot analysis of fresh frozen tissues. Genotyping of previously published alleles of the FKBP5 gene was also performed. We found correlation of upregulation of both immunophilins in the HIV-infected groups. In the HIV+ population with MDD, FKBP4 expression is significantly higher while FKBP5 is more variable. After analyzing the FKBP5 gene for single nucleotide polymorphisms, we found that rs3800373 CC genotype is more frequent in the MDD and MDD/Psychosis groups. We hypothesized that the levels of FKBP51, as modulator of the nuclear translocation of GR, would be lower in MDD. Instead, an increase in FKBP51 at both the transcript (FKBP5) and protein level correlated with MDD. Increased FKBP4 expression of correlated to HIV+MDD but not to HIV without MDD.

Evidence suggests that increased glucocorticoid receptor (GR) signaling may contribute to cognitive decline with age. We hypothesized that alterations in GR signaling pathway molecules, FK506 binding protein (FKBP) 51 and FKBP52, were associated with memory impairment in aged mice. We used the single-trial object recognition test to measure short-term memory in 18 aged mice compared to 22 young mice, and employed quantitative immunohistochemistry to assess cellular expression of those three proteins in the frontal cortex, hippocampal CA1, and dentate gyrus. Values of the discrimination ratio (DR, a measure of novelty preference) in aged mice were significantly lower than those in young mice (mean 0.54 vs. 0.67, p = 0.003, t test). Aged mice with DR below 0.54 were considered impaired (n = 9). In the three neuroanatomic regions studied, the immunoreactivity normalized to the area measured (IRn) for GR was significantly increased in aged mice regardless of their task performance compared to young mice (p < 0.005), as was the FKBP52 IRn (p < 0.007, U test). In the frontal cortex and CA1, the FKBP51 IRn was significantly lower in impaired aged mice than in unimpaired aged mice (p < 0.01 and <0.05, respectively) and in young mice (p < 0.05 and <0.01, respectively, Dunn’s post hoc test). In aged mice, the frontal cortex FKBP51 IRn correlated directly with DR (rs = 0.68, p = 0.002, Spearman rank correlation). These observations suggest that recognition memory impairment in aged mice is associated with decreased FKBP51 expression that may promote GR-mediated glucocorticoid signaling to a greater extent than in unimpaired aged mice.