Downregulation of myelination, energy, and translational genes in Menkes disease brain

Menkes disease (MD) is an X-linked recessive neurodegenerative disorder caused by mutations in a copper-transporting p-type ATPase (ATP7A) that normally delivers copper to the central nervous system. The precise reasons for neurodegeneration in MD are poorly understood. We hypothesized that gene expression changes in a MD patient with a lethal ATP7A mutation would indicate pathophysiological cascades relevant to the effects of copper deficiency in the developing brain. To test this hypothesis, oligonucleotide probes for 12,000 genes arrayed on Affymetrix Human Genome U95 GeneChips were used for expression profiling of fluorescently labeled primary cRNAs from post-mortem cerebral cortex and cerebellum of a MD patient who died at 6 months of age and a normal control brain matched for age, gender, and race. Histopathologic analysis of the proband's brain showed preservation of neuronal integrity and no hypoxic effects. However, cerebrospinal fluid and brain copper levels were subnormal, and expression profiling identified over 350 known dysregulated genes. For a subset of genes (approximately 12%) analyzed by quantitative RT-PCR, the correct cross-validation rate was 88%. Thirty known genes were altered in both cortex and cerebellum. Downregulation of genes involved in myelination, energy metabolism, and translation was the major finding. The cerebellum was more sensitive to copper deficiency.     

Abnormal HNK-1 expression in the cerebellum of an N-CAM null mouse

Human natural killer antigen-1 (HNK-1) is a carbohydrate epitope associated with sulfoglucuronylglycolipids and glycoproteins. Biochemical analyses have demonstrated associations between the HNK-1 epitope and isoforms of the neural cell adhesion molecule (N-CAM) family. In the cerebellum, HNK-1 is prominently expressed in Purkinje cell dendrites and Golgi cells. Purkinje cell expression of HNK-1 reveals an array of parasagittal stripes and transverse zones. Interestingly, the parasagittal expression pattern of HNK-1 is different from those reported with several other markers such as zebrin II/aldolase C and the small heat shock protein HSP25. N-CAM null knockout mice were used to explore the possible role of the HNK-1/N-CAM interaction during the topographical organization of the cerebellar cortex. N-CAM null mice have no N-CAM immunoreactivity but otherwise the cerebellum appears morphologically normal. Further, in the N-CAM null HNK-1 immunoreactivity is abolished from Purkinje cell dendrites but is retained on Golgi cells and neurons of the cerebellar nuclei. Despite the absence of N-CAM/HNK-1, parasagittal stripes and transverse zones in the cerebellum as revealed by using zebrin II immunocytochemistry appear normal.     

Distribution of Rlim, an LIM homeodomain gene, in the rat brain

We examined the distribution of Rlim, a homologue to Xlim-1, in the rat brain. Rlim, a LIM class homeodomain gene, was isolated from rat brain, and localized in the adult brain by in situ hybridization histochemistry. The expression of Rlim was found in discrete regions, such as the Purkinje cell layer of the cerebellum and several nuclei of the hypothalamus, midbrain and pons. This suggests that Rlim is related to regulation of genes that are specific to some neurons such as Purkinje cells in the adult.     

Aging and classical conditioning: parallel studies in rabbits and humans

The model system of classical conditioning of the eyelid response in rabbits has led to significant progress in the understanding of the neural circuitry involved in learning and memory. We are now at a point where we can begin to explore how aging in the neural circuitry affects this basic form of associative learning and memory over the life span. Parallels between humans and animals in aging and classical conditioning of the eyelid response are identified. A hypothetical model describing how acquisition occurs in the cerebellum has proved useful in predicting where in the cerebellum age changes might be most likely to affect classical conditioning. The loss of Purkinje cells with age is one of the cerebellar age changes likely to affect classical conditioning in aged mammals. Purkinje cell loss is only one of several age changes in the cerebellum which may impair acquisition and retention of the conditioned eyeblink response. This model system is of demonstrated utility in extending understanding of the neurobiology of learning, memory, and aging in humans as well as animals.     

Glutathione S-transferase isoenzymatic response to aging in rat cerebral cortex and cerebellum

Aging is associated with increased oxidant generation. One mechanism involved in the defense of oxidative products is the family of glutathione transferases (GST). We have analyzed the activity, distribution and expression of GSTP1 and GSTA4 isoenzymes in the cerebral cortex and cerebellum of young, adult and aged rats. The total GST activity, measured with the universal substrate 1-chloro-2,4-dinitrobenzene (CDNB), increased only with the maturation process; however GSTA4 activity, using the specific substrate 4-hydroxynonenal (HNE), did show an age-dependent increase in both brain regions. Cellular location of GSTA4 in astrocytes was not changed except for young cerebral cortex and adult/aged cerebellum that also showed immunoreactivity in layer III pyramidal neurons and Bergman radial glia, respectively. Distribution of GSTP1 was similar among groups and only an increased number of positive oligodendrocytes was found in the Purkinje and granular layer of adult/aged cerebellum. The GSTA4 and GSTP1 expression increased from young to adult/aged brain and GSTA4 even augmented in the aged cerebral cortex. These results suggest a GST isoenzymatic response with aging, but above all with the maturation process.     

Deposition of hyperphosphorylated tau in cerebellum of PS1 E280A Alzheimer's disease

Early-onset familial Alzheimer's disease (AD) caused by presenilin-1 mutation E280A (PS1-E280A) presents wide clinical and neuropathological variabilities. We characterized clinically and neuropathologically PS1-E280A focusing in cerebellar involvement and compared it with early-onset sporadic Alzheimer's disease (EOSAD). Twelve E280A brains and 12 matched EOSAD brains were analyzed for beta-amyloid and hyperphosphorylated tau (pTau) morphology, beta-amyloid subspecies 1-40, 1-42 levels, pTau levels, and expression of stress kinases in frontal cortex and cerebellum. The data were correlated to clinical and genetic findings. We observed higher beta-amyloid load, beta-amyloid 1-42 and pTau concentrations in frontal cortex of PS1-E280A compared with EOSAD. High beta-amyloid load was found in the cerebellum of PS1-E280A and EOSAD patients. In PS1-E280A, beta-amyloid localized to the molecular and Purkinje cell layers, whereas EOSAD showed them in Purkinje and granular cell layers. Surprisingly, 11 out of 12 PS1-E280A patients showed deposition of pTau in the cerebellum. Also, seven out of 12 PS1-E280A patients presented cerebellar ataxia. We conclude that deposition of beta-amyloid in the cerebellum is prominent in early-onset AD irrespective of genetic or sporadic origin. The presence of pTau in cerebellum in PS1-E280A underscores the relevance of cerebellar involvement in AD and might be correlated to clinical phenotype.     

Genome-wide analysis of miRNA expression reveals a potential role for miR-144 in brain aging and spinocerebellar ataxia pathogenesis

Neurodegenerative pathologies associated with aging exhibit clinical and morphological features that are relatively specific to humans. To gain insights into the evolution of the regulatory mechanisms of the aged brain, we compared age-related differences in microRNA (miRNA) expression levels in the cortex and cerebellum of humans, chimpanzees and rhesus macaques on a genome-wide scale. In contrast to global miRNA downregulation, a small subset of miRNAs was found to be selectively upregulated in the aging brain of all 3 species. Notably, miR-144 that is highly conserved appeared to be associated with the aging progression. Moreover, miR-144 plays a central role in regulating the expression of ataxin 1 (ATXN1), the disease-causing gene for the development spinocerebellar ataxia type 1 (SCA1). miRNA activity, including miR-144, -101 and -130 processing, was increased in the cerebellum and cortex of SCA1 and Alzheimer patients relative to healthy aged brains. Importantly, miR-144 and -101 inhibition increased ATXN1 levels in human cells. Thus, the activation of miRNA expression in the aging brain may serve to reduce the cytotoxic effect of polyglutamine expanded ATXN1 and the deregulation of miRNA expression may be a risk factor for disease development.     

Microglial activation in healthy aging

Healthy brain aging is characterized by neuronal loss and decline of cognitive function. Neuronal loss is closely associated with microglial activation and postmortem studies have indeed suggested that activated microglia may be present in the aging brain. Microglial activation can be quantified in vivo using (R)-[(11)C]PK11195 and positron emission tomography. The purpose of this study was to measure specific binding of (R)-[(11)C]PK11195 in healthy subjects over a wide age range. Thirty-five healthy subjects (age range 19-79 years) were included. In all subjects 60-minute dynamic (R)-[(11)C]PK11195 scans were acquired. Specific binding of (R)-[(11)C]PK11195 was calculated using receptor parametric mapping in combination with supervised cluster analysis to extract the reference tissue input function. Increased binding of (R)-[(11)C]PK11195 with aging was found in frontal lobe, anterior and posterior cingulate cortex, medial inferior temporal lobe, insula, hippocampus, entorhinal cortex, thalamus, parietal and occipital lobes, and cerebellum. This indicates that activated microglia appear in several cortical and subcortical areas during healthy aging, suggesting widespread neuronal loss.     

Expression of Alcoholism-Relevant Genes in the Liver Are Differently Correlated to Different Parts of the Brain

The purpose of this study is to investigate whether expression profiles of alcoholism-relevant genes in different parts of the brain are correlated differently with those in the liver. Four experiments were conducted. First, we used gene expression profiles from five parts of the brain (striatum, prefrontal cortex, nucleus accumbens, hippocampus, and cerebellum) and from liver in a population of recombinant inbred mouse strains to examine the expression association of 10 alcoholism-relevant genes. Second, we conducted the same association analysis between brain structures and the lung. Third, using five randomly selected, nonalcoholism-relevant genes, we conducted the association analysis between brain and liver. Finally, we compared the expression of 10 alcoholism-relevant genes in hippocampus and cerebellum between an alcohol preference strain and a wild-type control. We observed a difference in correlation patterns in expression levels of 10 alcoholism-relevant genes between different parts of the brain with those of liver. We then examined the association of gene expression between alcohol dehydrogenases (Adh1, Adh2, Adh5, and Adh7) and different parts of the brain. The results were similar to those of the 10 genes. Then, we found that the association of those genes between brain structures and lung was different from that of liver. Next, we found that the association patterns of five alcoholism-nonrelevant genes were different from those of 10 alcoholism-relevant genes. Finally, we found that the expression level of 10 alcohol-relevant genes is influenced more in hippocampus than in cerebellum in the alcohol preference strain. Our results show that the expression of alcoholism-relevant genes in liver is differently associated with the expression of genes in different parts of the brain. Because different structural changes in different parts of the brain in alcoholism have been reported, it is important to investigate whether those structural differences in the brains of those with alcoholism are due to the difference in the associations of gene expression between genes in liver and in different parts of the brain.     

Gene expression profiling of aging using DNA microarrays.

We have previously employed high density oligonucleotide arrays representing thousands of genes to determine the gene expression profile of the aging process in skeletal muscle (gastrocnemius) and brain (cerebellum and neocortex) of male C57BL/6 mice. Specific gene expression profiles are associated with the aging process of individual organs, and caloric restriction can prevent or retard the establishment of these gene expression alterations. The use of DNA microarrays may provide a new tool to measure biological age on a tissue-specific basis and to evaluate at the molecular level the efficacy of interventions designed to retard the aging process.     

Golgi-Kopsch silver study of the brain of a patient with untreated phenylketonuria, seizures, and cortical blindness.

This report describes the morphological changes observed in the brain of an untreated 27-year-old man with phenylketonuria, cortical blindness, and seizures. Golgi-Kopsch silver, cresyl violet, and hematoxylin and eosin stains were used to study cell structure and organization of the cerebellum, the lateral geniculate nuclei, the visual cortex, frontal cortex, and hippocampus. Extensive neuronal losses occurred in the right lateral geniculate nucleus (LGN), the visual cortex, and hippocampus. The left LGN, cerebellum, and frontal cortex retained neuronal components; there was a reduction in the number of dendritic processes on the Purkinje cells of the PKU subject. The loss of neurons in the LGN and occipital cortex is related to the blindness and the neuronal loss in the hippocampus is related to seizure activity.     

Neuroinflammatory phenotype in early Alzheimer's disease

Alzheimer's disease (AD) involves progressive neurodegeneration in the presence of misfolded proteins and poorly-understood inflammatory changes. However, research has shown that AD is genetically, clinically, and pathologically heterogeneous. In frozen brain samples of frontal cortex (diseased) and cerebellum (nondiseased) from the University of Kentucky Alzheimer's Disease Center autopsy cohort, we performed gene expression analysis for genes categorizing inflammatory states (termed M1 and M2) from early and late stage AD, and age-matched nondemented controls. We performed analysis of the serum samples for a profile of inflammatory proteins and examined the neuropathologic data on these samples. Striking heterogeneity was found in early AD. Specifically, early-stage AD brain samples indicated apparent polarization toward either the M1 or M2 brain inflammatory states when compared with age-matched nondisease control tissue. This polarization was observed in the frontal cortex and not in cerebellar tissue. We were able to detect differences in AD neuropathology, and changes in serum proteins that distinguished the individuals with apparent M1 versus M2 brain inflammatory polarization.     

Golgi-Kopsch silver study of the brain of a patient with untreated phenylketonuria,seizures, and cortical blindness

This report describes the morphological changes observed in the brain of an untreated 27-year-old man with phenylketonuria, cortical blindness, and seizures. Golgi–Kopsch silver, cresyl violet, and hematoxylin and eosin stains were used to study cell structure and organization of the cerebellum, the lateral geniculate nuclei, the visual cortex, frontal cortex, and hippocampus. Extensive neuronal losses occurred in the right lateral geniculate nucleus (LGN), the visual cortex, and hippocampus. The left LGN, cerebellum, and frontal cortex retained neuronal components; there was a reduction in the number of dendritic processes on the Purkinje cells of the PKU subject. The loss of neurons in the LGN and occipital cortex is related to the blindness and the neuronal loss in the hippocampus is related to seizure activity. © Wiley-Liss, Inc.     

Involvement of neurotrophic factors in aging of noradrenergic innervations in hippocampus and frontal cortex

In the present study, we investigated the age-dependent changes in the axon terminals of the locus coeruleus (LC) neurons in the frontal cortex and hippocampus, in which a high degree of axonal branching in the middle-aged brain was suggested to occur in our previous electrophysiological study. We used 6-, 13- and 25-month-old male F344/N rats, and performed Western blot analysis of the norepinephrine transporter (NET), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). NET expression level increased in the 13-month-old hippocampus, but was not altered by aging in the frontal cortex. BDNF expression level increased in the hippocampus, but did not change with age in the frontal cortex. On the other hand, GDNF expression level was increased with age in the frontal cortex, but was not in the hippocampus. These results suggest that the LC noradrenergic innervations may be locally regulated by different neurotrophic factors that exert their trophic actions at different target sites.