Scott E. Counts

Cholinergic system during the progression of Alzheimer’s disease: therapeutic implications

Alzheimer’s disease (AD) is characterized by a progressive phenotypic downregulation of markers within cholinergic basal forebrain (CBF) neurons, frank CBF cell loss and reduced cortical choline acetyltransferase activity associated with cognitive decline. Delaying CBF neurodegeneration or minimizing its consequences is the mechanism of action for most currently available drug treatments for cognitive dysfunction in AD. Growing evidence suggests that imbalances in the expression of NGF, its precursor proNGF and the high (TrkA) and low (p75NTR) affinity NGF receptors are crucial factors underlying CBF dysfunction in AD. Drugs that maintain a homeostatic balance between TrkA and p75NTR may slow the onset of AD. A NGF gene therapy trial reduced cognitive decline and stimulated cholinergic fiber growth in humans with mild AD. Drugs treating the multiple pathologies and clinical symptoms in AD (e.g., M1 cholinoceptor and/or galaninergic drugs) should be considered for a more comprehensive treatment approach for cholinergic dysfunction.

Down regulation of trk but not p75NTR gene expression in single cholinergic basal forebrain neurons mark the progression of Alzheimer's disease.

Dysfunction of cholinergic basal forebrain (CBF) neurons of the nucleus basalis (NB) is a cardinal feature of Alzheimers disease (AD) and correlates with cognitive decline. Survival of CBF neurons depends upon binding of nerve growth factor (NGF) with high‐affinity (trkA) and low‐affinity (p75NTR) neurotrophin receptors produced within CBF neurons. Since trkA and p75NTR protein levels are reduced within CBF neurons of people with mild cognitive impairment (MCI) and mild AD, trkA and/or p75NTR gene expression deficits may drive NB degeneration. Using single cell expression profiling methods coupled with custom‐designed cDNA arrays and validation with real‐time quantitative PCR (qPCR) and in situ hybridization, individual cholinergic NB neurons displayed a significant down regulation of trkA, trkB, and trkC expression during the progression of AD. An intermediate reduction was observed in MCI, with the greatest decrement in mild to moderate AD as compared to controls. Importantly, trk down regulation is associated with cognitive decline measured by the Global Cognitive Score (GCS) and the Mini‐Mental State Examination (MMSE). In contrast, there is a lack of regulation of p75NTR expression. Thus, trk defects may be a molecular marker for the transition from no cognitive impairment (NCI) to MCI, and from MCI to frank AD.

Reduction of cortical TrkA but not p75NTR protein in early‐stage Alzheimer's disease

Degeneration of cholinergic nucleus basalis (NB) cortical projection neurons is associated with cognitive decline in late‐stage Alzheimers disease (AD). NB neuron survival is dependent on coexpression of the nerve growth factor (NGF) receptors p75NTR and TrkA, which bind NGF in cortical projection sites. We have shown previously a significant reduction of NB perikarya expressing p75NTR and TrkA protein during the early stages of AD. Whether there is a concomitant reduction in cortical levels of these receptors during the progression of AD is unknown. p75NTR and TrkA protein was evaluated by quantitative immunoblotting in five cortical regions (anterior cingulate, superior frontal, superior temporal, inferior parietal, and visual cortex) of individuals clinically diagnosed with no cognitive impairment (NCI), mild cognitive impairment (MCI), mild/moderate AD, or severe AD. Cortical p75NTR levels were stable across the diagnostic groups. In contrast, TrkA levels were reduced approximately 50% in mild/moderate and severe AD compared with NCI and MCI in all regions except visual cortex. Mini‐Mental Status Examination scores correlated with TrkA levels in anterior cingulate, superior frontal, and superior temporal cortex. The selective reduction of cortical TrkA levels relative to p75NTR may have important consequences for cholinergic NB function during the transition from MCI to AD. Ann Neurol 2004

Differential expression of synaptic proteins in the frontal and temporal cortex of elderly subjects with mild cognitive impairment

Alterations in synaptic protein stoichiometry may contribute to neocortical synaptic dysfunction in Alzheimer disease (AD). Whether perturbations in synaptic protein expression occur during the earliest stages of cognitive decline remain unclear. We examined protein levels of synaptophysin (SYP), synaptotagmin (SYT), and drebrin (DRB) in 5 neocortical regions (anterior cingulate, superior frontal, superior temporal, inferior parietal, and visual) of people clinically diagnosed with no cognitive impairment (NCI), mild cognitive impairment (MCI), mild/moderate AD, or severe AD. Normalized SYP levels were decreased approximately 35% in the superior temporal and inferior parietal cortex in severe AD compared with NCI. SYT levels were unchanged across clinical diagnosis in the cortical regions. Levels of DRB, a dendritic spine plasticity marker, were reduced approximately 40% to 60% in all cortical regions in AD compared with NCI. DRB protein was also reduced approximately 35% in the superior temporal cortex of MCI subjects, and DRB and SYP levels in the superior temporal cortex correlated with Mini-Mental State Examination and Braak scores. In contrast, DRB levels in the superior frontal cortex increased approximately 30% in MCI subjects. The differential changes in DRB expression in the frontal and temporal cortex in MCI suggest a disparity of dendritic plasticity within these regions that may contribute to the early impairment of temporal cortical functions subserving memory and language compared with the relative preservation of frontal cortical executive function during the initial stages of cognitive decline.

Microarray analysis of hippocampal CA1 neurons implicates early endosomal dysfunction during Alzheimer’s disease progression

BACKGROUND Endocytic dysfunction and neurotrophin signaling deficits may underlie the selective vulnerability of hippocampal neurons during the progression of Alzheimers disease (AD), although there is little direct in vivo and biochemical evidence to support this hypothesis. METHODS Microarray analysis of hippocampal CA1 pyramidal neurons acquired via laser capture microdissection was performed using postmortem brain tissue. Validation was achieved using real-time quantitative polymerase chain reaction and immunoblot analysis. Mechanistic studies were performed using human fibroblasts subjected to overexpression with viral vectors or knockdown via small interference RNA. RESULTS Expression levels of genes regulating early endosomes (rab5) and late endosomes (rab7) are selectively upregulated in homogeneous populations of CA1 neurons from individuals with mild cognitive impairment and AD. The levels of these genes are selectively increased as antemortem measures of cognition decline during AD progression. Hippocampal quantitative polymerase chain reaction and immunoblot analyses confirmed increased levels of these transcripts and their respective protein products. Elevation of select rab GTPases regulating endocytosis paralleled the downregulation of genes encoding the neurotrophin receptors TrkB and TrkC. Overexpression of rab5 in cells suppressed TrkB expression, whereas knockdown of TrkB expression did not alter rab5 levels, suggesting that TrkB downregulation is a consequence of endosomal dysfunction associated with elevated rab5 levels in early AD. CONCLUSIONS These data support the hypothesis that neuronal endosomal dysfunction is associated with preclinical AD. Increased endocytic pathway activity, driven by elevated rab GTPase expression, may result in long-term deficits in hippocampal neurotrophic signaling and represent a key pathogenic mechanism underlying AD progression.

Gene Expression Profiles of Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

Cholinergic neurons of the nucleus basalis (NB) are selectively vulnerable in Alzheimers disease (AD), yet the molecular mechanisms associated with their dysfunction remain unknown. We used single cell RNA amplification and custom array technology to examine the expression of functional classes of mRNAs found in anterior NB neurons from normal aged and AD subjects. mRNAs encoding neurotrophin receptors, synaptic proteins, protein phosphatases, and amyloid-related proteins were evaluated. We found that trkB and trkC mRNAs were selectively down-regulated in NB neurons, whereas p75NTR mRNA levels remained stable in end stage AD. TrkA mRNA was reduced by approximately 28%, but did not reach statistical significance. There was a down-regulation of synaptophysin, synaptotagmin, and protein phosphatases PP1α and PP1β mRNAs in AD. In contrast, we found a selective up-regulation of cathepsin D mRNA in NB neurons in AD brain. Thus, anterior NB neurons undergo selective alterations in gene expression in AD. These results may provide clues to the molecular pathogenesis of NB neuronal degeneration during AD.

Mild cognitive impairment: pathology and mechanisms

Mild cognitive impairment (MCI) is rapidly becoming one of the most common clinical manifestations affecting the elderly. The pathologic and molecular substrate of people diagnosed with MCI is not well established. Since MCI is a human specific disorder and neither the clinical nor the neuropathological course appears to follow a direct linear path, it is imperative to characterize neuropathology changes in the brains of people who came to autopsy with a well-characterized clinical diagnosis of MCI. Herein, we discuss findings derived from clinical pathologic studies of autopsy cases who died with a clinical diagnosis of MCI. The heterogeneity of clinical MCI imparts significant challenges to any review of this subject. The pathologic substrate of MCI is equally complex and must take into account not only conventional plaque and tangle pathology but also a wide range of cellular, biochemical and molecular deficits, many of which relate to cognitive decline as well as compensatory responses to the progressive disease process. The multifaceted nature of the neuronal disconnection syndrome associated with MCI suggests that there is no single event which precipitates this prodromal stage of AD. In fact, it can be argued that neuronal degeneration initiated at different levels of the central nervous system drives cognitive decline as a final common pathway at this stage of the dementing disease process.

Single-cell gene expression analysis: implications for neurodegenerative and neuropsychiatric disorders.

Technical and experimental advances in microaspiration techniques, RNA amplification, quantitative real-time polymerase chain reaction (qPCR), and cDNA microarray analysis have led to an increase in the number of studies of single-cell gene expression. In particular, the central nervous system (CNS) is an ideal structure to apply single-cell gene expression paradigms. Unlike an organ that is composed of one principal cell type, the brain contains a constellation of neuronal and noneuronal populations of cells. A goal is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and noneuronal cells. The unprecedented resolution afforded by single-cell RNA analysis in combination with cDNA microarrays and qPCR-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease states. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models as well as postmortem human brain tissues. This focused review illustrates the potential power of single-cell gene expression studies within the CNS in relation to neurodegenerative and neuropsychiatric disorders such as Alzheimers disease (AD) and schizophrenia, respectively.

Neuropathology of mice carrying mutant APP(swe) and/or PS1(M146L) transgenes : Alterations in the p75(NTR) cholinergic basal forebrain septohippocampal pathway

Cholinergic basal forebrain (CBF) projection systems are defective in late Alzheimers disease (AD). We examined the brains of 12-month-old singly and doubly transgenic mice overexpressing mutant amyloid precursor protein (APP(swe)) and/or presenilin-1 (PS1(M146L)) to investigate the effects of these AD-related genes on plaque and tangle pathology, astrocytic expression, and the CBF projection system. Two types of beta-amyloid (Abeta)-immunoreactive (ir) plaques were observed: type 1 were darkly stained oval and elongated deposits of Abeta, and type 2 were diffuse plaques containing amyloid fibrils. APP(swe) and PS1(M146L) mouse brains contained some type 1 plaques, while the doubly transgenic (APP(swe)/PS1(M146L)) mice displayed a greater abundance of types 1 and 2 plaques. Sections immunostained for the p75 NGF receptor (p75(NTR)) revealed circular patches scattered throughout the cortex and hippocampus of the APP(swe)/PS1(M146L) mice that contained Abeta, were innervated by p75(NTR)-ir neurites, but displayed virtually no immunopositive neurons. Tau pathology was not seen in any transgenic genotype, although a massive glial response occurred in the APP(swe)/PS1(M146L) mice associated with amyloid plaques. Stereology revealed a significant increase in p75(NTR)-ir medial septal neurons in the APP(swe) and PS1(M146L) singly transgenic mice compared to the APP(swe)/PS1(M146L) mice. No differences in size or optical density of p75(NTR)-ir neurons were observed in these three mutants. p75(NTR)-ir fibers in hippocampus and cortex were more pronounced in the APP(swe) and PS1(M146L) mice, while the APP(swe)/PS1(M146L) mice showed the least p75(NTR)-ir fiber staining. These findings suggest a neurotrophic role for mutant APP and PS1 upon cholinergic hippocampal projection neurons at 12 months of age.

Noradrenaline activation of neurotrophic pathways protects against neuronal amyloid toxicity

J. Neurochem. (2010) 113, 649–660.