Khalid Iqbal

STALOSYLGALACTOSYL CERAMTDE AS A SPECIFIC MARKER FOR HUMAN MYELIN AND OLIGODENDROGLIAL PERIKARYA: GANGLIOSIDES OF HUMAN MYELIN, OLIGODENDROGLIA AND NEURONS

Gangliosides were isolated from human brain myelin, oligodendroglia, and neurons. Quantitative analysis revealed the following ganglioside contents: myelin, 2.0; neurons, 1.3; and oligodendroglia, 0.35 μg ganglioside sialic acid per mg protein. Myclin had a relatively simple ganglioside pattern with GM4 and GM1 as the predominant ganglioside species. The ganglioside pattern of oligodendroglia was quite complex and it resembled that of whole white matter rather than that of myelin. A high concentration of GM4 was found in oligodendroglial fractions in addition to GM1, GD1a, GD1b, and GT1b. The usually‐ minor brain gangliosides GM3, GM2, and GM3 were also enriched in oligodendroglia. The neuronal ganglioside pattern was generally similar to the pattern of whole gray matter. Both neurons and whole gray matter contained very low amounts of GM4. These results indicate that GM4 is specifically localized in myelin and oligodendroglia of the CNS. Evidence is also presented that myelin, but not oligodendroglia, is the major reservoir of human white matter GM1 and GM4.

Mechanisms of neurofibrillary degeneration and the formation of neurofibrillary tangles

Alzheimer disease (AD) has polyetiology. Independent of the etiology the disease is characterized histopathologically by the intraneuronal accumulation of paired helical filaments (PHF), forming neurofibrillary tangles, neuropil threads and dystrophic neurites surrounding the extracellular deposits of beta-amyloid in plaques, the second major lesion. The clincal expression of AD correlates with the presence of neurofibrillary degeneration; beta-amyloid alone does not produce the disease clinically. Thus arresting neurofibrillary degeneration offers a promising key target for therapeutic intervention of AD. The major protein subunit of PHF is the microtubule-associated protein tau. Tau in AD brain, especially PHF, is abnormally hyperphosphorylated and glycosylated. With maturation, the tangles are increasingly ubiquitinated. Levels of tau and conjugated ubiquitin are elevated both in AD brain and CSF. The AD abnormally phosphorylated tau (AD P-tau) does not promote microtubule assembly, but on dephosphorylation its microtubule promoting activity is restored to approximately that of the normal tau. The AD P-tau competes with tubulin in binding to normal tau, MAP1 and MAP2 and inhibits their microtubule assembly promoting activities. Furthermore, the AD P-tau sequesters normal MAPs from microtubules. The association of AD P-tau with normal tau but not with MAP1 or MAP2 results in the formation of tangles of 3.3 +/- 0.5 mm filaments. Deglycosylation of Alzheimer neurofibrillary tangles with endoglycosidase F/N-glycosidase F untwists the PHF resulting in tangles of thin filaments similar to those formed by association between the AD P-tau and normal tau. Dephosphorylation or deglycosylation plus dephosphorylation but not deglycosylation alone restores the microtubule assembly promoting activity of tau. In vitro AD P-tau can be dephosphorylated by protein phosphatases PP-2B, PP-2A and PP-1 but not PP-2C and all the three tau phosphatases are present in brain neurons. Tau phosphatase activity is decreased by approximately 30% in AD brain. Inhibition of PP-2A and PP-1 activities in SY5Y neuroblastoma by 10 nM okadaic acid causes breakdown of microtubules and the degeneration of these cells. It is suggested (I) that a defect(s) in the protein phosphorylation/dephosphorylation system(s) leads to a hyperphosphorylation of tau, (ii) that this altered tau causes disassembly of microtubules and consequently a retrograde neuronal degeneration; (iii) a pharmacological approach to AD is to enhance the tau phosphatase activity; and (iv) that CSF tau and conjugated ubiquitin levels are promising markers of AD brain pathology.

Neurofibrillary and Synaptic Pathology in the Aged Brain

Normal aging in the human brain may be thought of as a state in which pathological alterations exist without obvious clinical expression. Unlike other organs in which there is a repetition of structural-functional units, the brain is a complex collection of groups of nerve cells, each with varying metabolic and functional characteristics (a multiorgan organ). A decline in the number of brain cells or key connections with one another, when below a critical reserve level, would be expected to result in a deterioration of function, and create difficulties in coping with additional noxious or infectious stress. At the same time, genetically programmed time-associated changes in aging brain cells may increase their susceptibility to harmful environmental effects (hormonal, infectious, immunological) and lead to various pathological changes found in the aged brain.

Microtubule associated protein tau binds to double-stranded but not single-stranded DNA.

Abstract. Tau, a major microtubule-associated protein of the neuron, which is known to promote the assembly of and to stabilize microtubules, has also been seen associated with chromatin in neuronal cell lines, but its role in this subcellular compartment is still unknown. In this study, the binding of tau to DNA was investigated using the electrophoretic mobility shift assay. Using polynucleotide as probe, we found that tau bound to double-stranded but not to single-stranded DNA. Formation of tau-polynucleotide complex was disrupted by alkaline pH and a high concentration of NaCl, but was not affected by dithiothreitol. Electron microscopy revealed that the protein associated with the nucleic acid in a necklacelike manner. DNA-cellulose chromatography and radioimmunodot-blot analyses showed that calf thymus histones VI-S, VII-S and VIII-S could replace both recombinant human brain tau352 (tau-23) and tau441 (tau-40) from DNA. Thus, tau appears to bind to DNA reversibly in the presence of histones.

Neurotrophic factor small-molecule mimetics mediated neuroregeneration and synaptic repair: emerging therapeutic modality for Alzheimer's disease.

Alzheimer’s disease (AD) is an incurable and debilitating chronic progressive neurodegenerative disorder which is the leading cause of dementia worldwide. AD is a heterogeneous and multifactorial disorder, histopathologically characterized by the presence of amyloid β (Aβ) plaques and neurofibrillary tangles composed of Aβ peptides and abnormally hyperphosphorylated tau protein, respectively. Independent of the various etiopathogenic mechanisms, neurodegeneration is a final common outcome of AD neuropathology. Synaptic loss is a better correlate of cognitive impairment in AD than Aβ or tau pathologies. Thus a highly promising therapeutic strategy for AD is to shift the balance from neurodegeneration to neuroregeneration and synaptic repair. Neurotrophic factors, by virtue of their neurogenic and neurotrophic activities, have potential for the treatment of AD. However, the clinical therapeutic usage of recombinant neurotrophic factors is limited because of the insurmountable hurdles of unfavorable pharmacokinetic properties, poor blood–brain barrier (BBB) permeability, and severe adverse effects. Neurotrophic factor small-molecule mimetics, in this context, represent a potential strategy to overcome these short comings, and have shown promise in preclinical studies. Neurotrophic factor small-molecule mimetics have been the focus of intense research in recent years for AD drug development. Here, we review the relevant literature regarding the therapeutic beneficial effect of neurotrophic factors in AD, and then discuss the recent status of research regarding the neurotrophic factor small-molecule mimetics as therapeutic candidates for AD. Lastly, we summarize the preclinical studies with a ciliary neurotrophic factor (CNTF) small-molecule peptide mimetic, Peptide 021 (P021). P021 is a neurogenic and neurotrophic compound which enhances dentate gyrus neurogenesis and memory processes via inhibiting leukemia inhibitory factor (LIF) signaling pathway and increasing brain-derived neurotrophic factor (BDNF) expression. It robustly inhibits tau abnormal hyperphosphorylation via increased BDNF mediated decrease in glycogen synthase kinase-3β (GSK-3β, major tau kinase) activity. P021 is a small molecular weight, BBB permeable compound with suitable pharmacokinetics for oral administration, and without adverse effects associated with native CNTF or BDNF molecule. P021 has shown beneficial therapeutic effect in several preclinical studies and has emerged as a highly promising compound for AD drug development.

Pharmacological targets to inhibit alzheimer neurofibrillary degeneration

Neurofibrillary degeneration appears to be required for the clinical expression of Alzheimer disease (AD) and related tauopathies. Given the polyetiology of these diseases and the pivotal involvement of neurofibrillary degeneration in their pathogenesis, inhibition of this lesion offers a promising therapeutic target. Studies from our laboratories have shown that there is a protein phosphorylation/dephosphorylation imbalance and that the microtubule associated protein tau is abnormally hyperphosphorylated in the brain of patients with AD and in this form it is the major protein subunit of paired helical filaments/neurofibrillary tangles (PHF/NFT). The abnormal tau which is polymerized into PHF/NFT neither promotes or inhibits in vitro microtubule assembly. In contrast the cytosolic abnormally hyperphosphorylated tau from AD brain, the AD P-tau neither associates with tubulin nor promotes in vitro microtubule assembly but instead it sequesters normal tau, MAP1 and MAP2 and inhibits microtubule assembly. The AD P-tau readily self-assembles in vitro into tangles of PHF/straight filaments under physiological conditions of protein concentration, pH, ionic strength and reducing conditions and this self assembly requires the abnormal hyperphosphorylation of this protein. The activity of phosphoseryl/phosphothreonyl protein phosphatase (PP)-2A which regulates the phosphorylation of tau, is compromised in AD brain. Thus, modulation of the activities of protein phosphatase-2A and tau kinases and inhibition of the sequestration of normal MAPs by AD P-tau offer promising therapeutic opportunities to inhibit neurofibrillary degeneration and the diseases characterized by this lesion.

Sera from Children with Autism Induce Autistic Features Which Can Be Rescued with a CNTF Small Peptide Mimetic in Rats

Autism is a neurodevelopmental disorder characterized clinically by impairments in social interaction and verbal and non-verbal communication skills as well as restricted interests and repetitive behavior. It has been hypothesized that altered brain environment including an imbalance in neurotrophic support during early development contributes to the pathophysiology of autism. Here we report that sera from children with autism which exhibited abnormal levels of various neurotrophic factors induced cell death and oxidative stress in mouse primary cultured cortical neurons. The effects of sera from autistic children were rescued by pre-treatment with a ciliary neurotrophic factor (CNTF) small peptide mimetic, Peptide 6 (P6), which was previously shown to exert its neuroprotective effect by modulating CNTF/JAK/STAT pathway and LIF signaling and by enhancing brain derived neurotrophic factor (BDNF) expression. Similar neurotoxic effects and neuroinflammation were observed in young Wistar rats injected intracerebroventricularly with autism sera within hours after birth. The autism sera injected rats demonstrated developmental delay and deficits in social communication, interaction, and novelty. Both the neurobiological changes and the behavioral autistic phenotype were ameliorated by P6 treatment. These findings implicate the involvement of neurotrophic imbalance during early brain development in the pathophysiology of autism and a proof of principle of P6 as a potential therapeutic strategy for autism.

Early-Onset Network Hyperexcitability in Presymptomatic Alzheimer’s Disease Transgenic Mice Is Suppressed by Passive Immunization with Anti-Human APP/Aβ Antibody and by mGluR5 Blockade

Cortical and hippocampal network hyperexcitability appears to be an early event in Alzheimer’s disease (AD) pathogenesis, and may contribute to memory impairment. It remains unclear if network hyperexcitability precedes memory impairment in mouse models of AD and what are the underlying cellular mechanisms. We thus evaluated seizure susceptibility and hippocampal network hyperexcitability at ~3 weeks of age [prior to amyloid beta (Aβ) plaque deposition, neurofibrillary pathology, and cognitive impairment] in a triple transgenic mouse model of familial AD (3xTg-AD mouse) that harbors mutated human Aβ precursor protein (APP), tau and presenilin 1 (PS1) genes. Audiogenic seizures were elicited in a higher proportion of 3xTg-AD mice compared with wild type (WT) controls. Seizure susceptibility in 3xTg-AD mice was attenuated either by passive immunization with anti-human APP/Aβ antibody (6E10) or by blockade of metabotropic glutamate receptor 5 (mGluR5) with the selective antagonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP). In in vitro hippocampal slices, suppression of synaptic inhibition with the GABAA receptor antagonist, bicuculline, induced prolonged epileptiform (>1.5 s in duration) ictal-like discharges in the CA3 neuronal network in the majority of the slices from 3xTg-AD mice. In contrast, only short epileptiform (<1.5 s in duration) interictal-like discharges were observed following bicuculline application in the CA3 region of WT slices. The ictal-like activity in CA3 region of the hippocampus was significantly reduced in the 6E10-immunized compared to the saline-treated 3xTg-AD mice. MPEP acutely suppressed the ictal-like discharges in 3xTg-AD slices. Remarkably, epileptiform discharge duration positively correlated with intraneuronal human (transgenic) APP/Aβ expression in the CA3 region of the hippocampus. Our data suggest that in a mouse model of familial AD, hypersynchronous network activity underlying seizure susceptibility precedes Aβ plaque pathology and memory impairment. This early-onset network hyperexcitability can be suppressed by passive immunization with an anti-human APP/Aβ antibody and by mGluR5 blockade in 3xTg-AD mice.

Alterations of the Neuronal Cytoskeleton in Alzheimer’s Disease and Related Conditions

The cytoskeleton of a normal mature neuron is composed of three types of fibrils, the microtubules, the neurofilaments and the microfilaments. One of the cellular and molecular changes with aging, the mechanism of which remains unknown to date, is the formation of argentophilic intracellular neurofibrillary tangles in certain selected neurons of the aged human brain. These extraordinary neurofibrillary changes are seen in great abundance in several adult and late life dementias especially the Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT) (for review see Iqbal, et al. 1977b; Iqbal and Wisniewski 1983). The Alzheimer neurofibrillary tangles (ANT) are composed of paired helical filaments (PHF). Bundles of these PHF are also found in neurites (Braak, et al. 1986), especially in the dystrophic neurites of the neuritic (senile) plaque, the second leading histopathological lesion of AD/SDAT. Together these two lesions, the ANT and the plaques, both of which contain the PHF, are the histopathological hallmark of AD/SDAT (Kidd, 1964; Terry, et al. 1964); occassionally either tangles of 15 nm straight filaments or these filaments admixed with PHF have been observed in a few AD/SDAT cases. (Shibayama and Kitoch, 1978; Yagishita, et. al. 1981). The number of ANT and plaques correlates positively with the degree of psychometric deficiency in the affected patients (Tomlinson, et. al. 1970), but their origin and role in disease are not understood.

Alzheimer Neurofibrillary Degeneration: A Feasible and Key Target for Therapeutics

Independent of the etiology, i.e. whether genetic or non-genetic, Alzheimer disease (AD) is characterized histopathologically by the intraneuronal accumulation of paired helical filaments (PHF), forming neurofibrillary tangles, neuropil threads and dystrophic neurites surrounding the extracellular deposits of β-amyloid, the second major lesion. The clinical expression of AD correlates with the presence of neurofibrillary degeneration; β-amyloidosis alone does not produce the disease clinically. Thus, arresting neurofibrillary degeneration offers a promising key target for therapeutic intervention of AD. In this chapter the molecular pathology, the mechanisms of neurofibrillary degeneration and therapeutic strategies to arrest this brain lesion in AD are briefly reviewed.