Proteomic Analysis of Adult Human Hippocampal Subfields Demonstrates Regional Heterogeneity in the Protein Expression.

Background: Distinct hippocampal subfields are known to get affected during aging, psychiatric disorders, and various neurological and neurodegenerative conditions. To understand the biological processes associated with each subfield, it is important to understand its heterogeneity at the molecular level. To address this lacuna, we investigated the proteomic analysis of hippocampal subfields─the cornu ammonis sectors (CA1, CA2, CA3, CA4) and dentate gyrus (DG) from healthy adult human cohorts. Findings: Microdissection of hippocampal subfields from archived formalin-fixed paraffin-embedded tissue sections followed by TMT-based multiplexed proteomic analysis resulted in the identification of 5,593 proteins. Out of these, 890 proteins were found to be differentially abundant among the subfields. Further bioinformatics analysis suggested proteins related to gene splicing, transportation, myelination, structural activity, and learning processes to be differentially abundant in DG, CA4, CA3, CA2, and CA1, respectively. A subset of proteins was selected for immunohistochemistry-based validation in an independent set of hippocampal samples. Conclusions: We believe that our findings will effectively pave the way for further analysis of the hippocampal subdivisions and provide awareness of its subfield-specific association to various neurofunctional anomalies in the future. The current mass spectrometry data is deposited and publicly made available through ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD029697.

Genetic diversity and proviral DNA load in different neural compartments of HIV-1 subtype C infection.

In India, the low prevalence of HIV-associated dementia (HAD) in the Human immunodeficiency virus type 1 (HIV-1) subtype C infection is quite paradoxical given the high-rate of macrophage infiltration into the brain. Whether the direct viral burden in individual brain compartments could be associated with the variability of the neurologic manifestations is controversial. To understand this paradox, we examined the proviral DNA load in nine different brain regions and three different peripheral tissues derived from ten human subjects at autopsy. Using a highly sensitive TaqMan probe-based real-time PCR, we determined the proviral load in multiple samples processed in parallel from each site. Unlike previously published reports, the present analysis identified uniform proviral distribution among the brain compartments examined without preferential accumulation of the DNA in any one of them. The overall viral DNA burden in the brain tissues was very low, approximately 1 viral integration per 1000 cells or less. In a subset of the tissue samples tested, the HIV DNA mostly existed in a free unintegrated form. The V3-V5 envelope sequences, demonstrated a brain-specific compartmentalization in four of the ten subjects and a phylogenetic overlap between the neural and non-neural compartments in three other subjects. The envelope sequences phylogenetically belonged to subtype C and the majority of them were R5 tropic. To the best of our knowledge, the present study represents the first analysis of the proviral burden in subtype C postmortem human brain tissues. Future studies should determine the presence of the viral antigens, the viral transcripts, and the proviral DNA, in parallel, in different brain compartments to shed more light on the significance of the viral burden on neurologic consequences of HIV infection.

Multiple NF-κB sites in HIV-1 subtype C long terminal repeat confer superior magnitude of transcription and thereby the enhanced viral predominance.

We demonstrate that at least three different promoter variant strains of HIV-1 subtype C have been gradually expanding and replacing the standard subtype C viruses in India, and possibly in South Africa and other global regions, over the past decade. The new viral strains contain an additional NF-κB, NF-κB-like, or RBEIII site in the viral promoter. Although the acquisition of an additional RBEIII site is a property shared by all the HIV-1 subtypes, acquiring an additional NF-κB site remains an exclusive property of subtype C. The acquired κB site is genetically distinct, binds the p50-p65 heterodimer, and strengthens the viral promoter at the levels of transcription initiation and elongation. The 4-κB viruses dominate the 3-κB "isogenic" viral strains in pairwise competition assays in T-cell lines, primary cells, and the ecotropic human immunodeficiency virus mouse model. The dominance of the 4-κB viral strains is also evident in the natural context when the subjects are coinfected with κB-variant viral strains. The mean plasma viral loads, but not CD4 counts, are significantly different in 4-κB infection suggesting that these newly emerging strains are probably more infectious. It is possible that higher plasma viral loads underlie selective transmission of the 4-κB viral strains. Several publications previously reported duplication or deletion of diverse transcription factor-binding sites in the viral promoter. Unlike previous reports, our study provides experimental evidence that the new viral strains gained a potential selective advantage as a consequence of the acquired transcription factor-binding sites and importantly that these strains have been expanding at the population level.

Quantitative proteomics for identifying biomarkers for Rabies.

INTRODUCTION: Rabies is a fatal acute viral disease of the central nervous system, which is a serious public health problem in Asian and African countries. Based on the clinical presentation, rabies can be classified into encephalitic (furious) or paralytic (numb) rabies. Early diagnosis of this disease is particularly important as rabies is invariably fatal if adequate post exposure prophylaxis is not administered immediately following the bite. METHODS: In this study, we carried out a quantitative proteomic analysis of the human brain tissue from cases of encephalitic and paralytic rabies along with normal human brain tissues using an 8-plex isobaric tags for relative and absolute quantification (iTRAQ) strategy. RESULTS AND CONCLUSION: We identified 402 proteins, of which a number of proteins were differentially expressed between encephalitic and paralytic rabies, including several novel proteins. The differentially expressed molecules included karyopherin alpha 4 (KPNA4), which was overexpressed only in paralytic rabies, calcium calmodulin dependent kinase 2 alpha (CAMK2A), which was upregulated in paralytic rabies group and glutamate ammonia ligase (GLUL), which was overexpressed in paralytic as well as encephalitic rabies. We validated two of the upregulated molecules, GLUL and CAMK2A, by dot blot assays and further validated CAMK2A by immunohistochemistry. These molecules need to be further investigated in body fluids such as cerebrospinal fluid in a larger cohort of rabies cases to determine their potential use as antemortem diagnostic biomarkers in rabies. This is the first study to systematically profile clinical subtypes of human rabies using an iTRAQ quantitative proteomics approach.

NMDA receptor activation by HIV-Tat protein is clade dependent.

In countries infected with HIV clade B, some patients develop a rapidly progressive dementia that if untreated results in death. In regions of the world infected with HIV clade C, only milder forms of cognitive impairment have been recognized. HIV-infected macrophages are the principal mediators of dementia. HIV clade C, however, efficiently infects macrophages and HIV-infected macrophages are found in the brains of clade C-infected patients. HIV-infected macrophages release Tat protein, which may act directly on neurons to cause toxicity. We found that Tat released from Tat-expressing cells was at least 1000-fold more toxic than recombinant Tat protein. We determined whether Tat could interact with NMDA receptors and whether these interactions are clade dependent. It is demonstrated that Tat binds directly to the NMDA receptor leading to excitotoxicity. The Cys 30-Cys 31 motif in Tat is critical for exciting the NMDA receptor and the Cys31Ser mutation found in clade C Tat has a significantly attenuated neurotoxic response. Through molecular modeling and site-directed mutagenesis, we predict that Cys 31 disrupts the disulfide bond between Cys 744 and Cys 798 on the NR1 subunit of the NMDA receptor by directly interacting with Cys 744 leading to a free thiol group on Cys 798 and subsequent persistent activation of the NMDA receptor.

An idiopathic epilepsy syndrome linked to 3q13.3-q21 and missense mutations in the extracellular calcium sensing receptor gene.

OBJECTIVE: To identify the disease locus in a three-generation south Indian family having several of its members affected with idiopathic epilepsy. METHODS: Genome-wide parametric linkage analysis was performed with 382 autosomal markers. Mutational analysis of the positional candidate genes in linked interval was performed by direct sequencing of genomic DNA from the proband in the family. Expression analysis in human adult brain was performed by Western blotting. RESULTS: A novel epilepsy genetic locus on chromosome 3q13.3-q21 was identified by linkage analysis. This locus comprises about 12 megabases of the genomic interval, with its proximal and distal genetic boundaries defined by microsatellite markers, D3S3675 and D3S1551, respectively. In this interval, we found a novel, patient-specific, missense variant, Arg898Gln, at the extracellular calcium sensing receptor (CASR), a gene belonging to the G-protein-coupled receptor family. CASR expression was detected in the temporal lobe, frontal lobe, parietal lobe, cerebellum, and hippocampus. Four additional, potentially pathogenic, missense CASR variants, Glu354Ala, Ile686Val, Ala988Val, and Ala988Gly, were observed in five individuals affected with idiopathic generalized epilepsy. INTERPRETATION: A novel idiopathic epilepsy locus has been mapped on chromosome 3q13.3-q21, as evident by presence of significant genetic linkage. Identification of novel, rare missense CASR variants at evolutionary-conserved residues in epilepsy patients and CASR expression in various subregions of human brain raises an interesting possibility of involvement of CASR in pathophysiology of epileptic disorders.

Exceptional molecular and coreceptor-requirement properties of molecular clones isolated from an Human Immunodeficiency Virus Type-1 subtype C infection.

BACKGROUND: The pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood. This situation is more complex in subtype C infection where coreceptor switch is either absent or extremely rare. To gain insights into the mechanisms that underlie coreceptor requirement of subtype C, we screened several primary viral isolates and identified a clinical sample that demonstrated a potential to grow on standard T-cell lines with no detectable CCR5 expression. The subject was diagnosed with HIV-1 associated dementia in the absence of opportunistic infections of the brain. To isolate molecular clones from this virus, we devised a novel strategy based on anchor primers that target a sequence in the reverse transcriptase, highly conserved among diverse subtypes of HIV-1. RESULTS: Using this strategy, we isolated 8 full-length molecular clones from the donor. Two of the eight molecular clones, 03In94_D17 and 03In94_D24, (D17 and D24) generated replication-competent viruses. Phylogenetic analysis of the full-length viral sequences revealed that both clones were non-recombinant subtype C viruses. They contain intact open reading frames in all the viral proteins. Both the viral clones are endowed with several unique molecular and biological properties. The viral promoter of the clones is characterized by the presence of four NF-kB binding elements, a feature rarely seen in the subtype C HIV-1 LTR. Interestingly, we identified the coexistence of two different forms of Rev, a truncated form common to subtype C and a full-length form less common for this subtype, in both proviral and plasma virus compartments. An exceptional property of the viruses, atypical of subtype C, is their ability to use a wide range of coreceptors including CCR5, CXCR4, and several others tested. Sequence analysis of Env of D17 and D24 clones identified differences within the variable loops providing important clues for the expanded coreceptor use. The V1, V2 and V4 loops in both of the molecular clones are longer due to the insertion of several amino acid residues that generated potential N-linked glycosylation sites. CONCLUSION: The exceptional biological and molecular properties of these clones make them invaluable tools to understand the unique pathogenic characteristics of subtype C.

Primary cardiac cytotoxic T-cell lymphoma presenting with neurological deficits: a case report.

BACKGROUND: Primary cardiac lymphoma is extremely rare in immunocompetent patients. Clinical manifestations vary, and, most often, diagnosis is not made until autopsy. The majority of reported primary cardiac lymphoma cases have been of B-cell origin, while T-cell cardiac lymphomas have been extremely rare. Occasionally, lymphomas and other systemic malignancies clinically present as paraneoplastic neurological syndromes. METHODS: We report a unique case of primary cardiac peripheral T-cell lymphoma of cytotoxic phenotype, clinically presenting with neurological features of external ophthalmoplegia and lower cranial nerve paresis mimicking mitochondrial cytopathy, that was recognized at autopsy. Brain and thoracoabdominal viscera retrieved at autopsy were fixed in 10% buffered formalin and processed for paraffin embedding. In addition to routine histology, immunohistochemistry for immunophenotypic characterization of lymphoma cells was performed. Fresh skeletal muscle was processed for cryosectioning and histochemical staining. RESULTS: On gross examination, the heart showed multiple circumscribed, whitish nodules on both sides. Histological examination of these nodules revealed lymphomatous deposits-cells expressing CD45, CD2, CD3, CD5, CD7, CD8, perforin, and granzyme B. Histological sections from the brain showed foci of demyelination and patchy perivascular lymphoid cell aggregates in leptomeninges and within the parenchyma. These lymphoid cells expressed CD2, CD3, and CD5, with the T cells being predominantly CD4 (CD4:CD8>2), which was unlike the CD8-predominant lymphomatous infiltrate in the heart. Hence, these lymphoid cells in the brain, rather than disseminated lymphoma cells, were considered to be related to the demyelinating process. There was no evidence of lymphomatous deposits in the rest of the viscera examined. CONCLUSION: A diagnosis of primary cardiac peripheral T-cell lymphoma of cytotoxic phenotype clinically manifesting as paraneoplastic demyelinating lesions in the brain was described.

Neuropathology of viral infections of the central nervous system.

Many viral infections of the nervous system cause stereotyped pathologic features and overlapping clinical and imaging features. Neuroimaging usually offers neuroanatomical localization of the pathology, degree of involvement of the nervous system, and response to therapy during follow up in a few instances. Neuroimaging is a useful adjunct for diagnosis.

Proteomic Analysis of the Human Anterior Pituitary Gland.

The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.

Characterization of human immunodeficiency virus (HIV)-infected cells in infiltrates associated with CNS opportunistic infections in patients with HIV clade C infection.

Infection with human immunodeficiency virus (HIV) clade C is the most common HIV infection worldwide, yet its impact on the nervous system remains largely unknown. Autopsy studies from regions affected by this virus are scarce, and HIV dementia has only rarely been reported from these countries. Most patients who develop neurologic complications die of opportunistic infections. We thus conducted a neuropathologic study from a single institution in India to characterize the HIV-infected cells in the inflammatory infiltrates in a total of 15 cases (5 patients each who died of either CNS toxoplasmosis, tuberculosis, or cryptococcal meningitis). Nearly, all patients had HIV-infected cells in the brain, although these cells were most abundant in patients with toxoplasma encephalitis. Interestingly, none of the patients had any multinucleated giant cells. HIV-infected cells were found in the parenchyma, perivascular regions, and choroid plexus and found infiltrating the parenchyma from the meninges, suggesting multiple portals of entry into the brain. These findings suggest the possibility that patients, even if successfully treated for an opportunistic inflection, may be at high risk of developing HIV encephalitis and subsequent dementia.

Gene expression analysis from human immunodeficiency virus type 1 subtype C promoter and construction of bicistronic reporter vectors.

We report the cloning and sequence analysis of the long terminal repeat (LTR) of several primary HIV-1 subtype C strains of India. Phylogenetically, all the LTRs and the paired env sequences clustered with subtype C reference strains. The LTRs demonstrated extensive polymorphism in the transcription factor binding sites (TFBS) within the enhancer and the modulator regions. We generated reporter vectors under the control of a select subset of the subtype C LTRs. The reporter vectors are distinguished by the simultaneous expression of two independent reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescence protein (EGFP), in response to Tat. Expression of EGFP was facilitated by engineering an internal ribosome entry site (IRES) into the expression cassette. Although subtype C strains cause a large majority of the global infections, and important differences in the transcription factor binding sites have been identified in the subtype C promoter, few reporter vectors containing subtype C-LTR have been described. We analyzed gene expression from the C-LTR reporter vectors in different cell lines under diverse experimental conditions and compared it to the B-LTR reporter vector. The reporter vectors were responsive to Tat derived from diverse viral subtypes. Furthermore, a positive correlation was observed between the expression of the reporter genes and the viral structural protein p24 when the cells were infected with viral molecular clones. The LTR reporters we developed could be of significant use in the study of viral transactivation, in the evaluation of biological properties of viral subtypes, and in the screening for antiviral inhibitors.

Characterization of human pineal gland proteome.

The pineal gland is a neuroendocrine gland located at the center of the brain. It is known to regulate various physiological functions in the body through secretion of the neurohormone melatonin. Comprehensive characterization of the human pineal gland proteome has not been undertaken to date. We employed a high-resolution mass spectrometry-based approach to characterize the proteome of the human pineal gland. A total of 5874 proteins were identified from the human pineal gland in this study. Of these, 5820 proteins were identified from the human pineal gland for the first time. Interestingly, 1136 proteins from the human pineal gland were found to contain a signal peptide domain, which indicates the secretory nature of these proteins. An unbiased global proteomic profile of this biomedically important organ should benefit molecular research to unravel the role of the pineal gland in neuropsychiatric and neurodegenerative diseases.

Identification of unique B/C recombinant strains of HIV-1 in the southern state of Karnataka, India.

We characterized the molecular nature of a large number of primary HIV-1 isolates in the four southern states of India. In addition to confirming a predominance of subtype C infection, for the first time we identified three B/C recombinant viruses in a subset of 115 samples. Unexpectedly, env sequences of two of the three B/C recombinants phylogenetically clustered with subtype B strains of the USA. The determination of the real incidence of the recombinant viruses is of importance.

Outcome of lesionectomy in medically refractory epilepsy due to non-mesial temporal sclerosis (non-MTS) lesions.

OBJECTIVES: To analyze the seizure outcome of lesionectomy for refractory epilepsy secondary to non-mesial temporal sclerosis (non-MTS) lesions. METHODS: Sixty-eight patients with non-MTS lesions (M:F=42:26; age at onset: 11.7±9.6 years; age at surgery: 21.1±9.4 years), who underwent lesionectomy for refractory epilepsy were analyzed. The age at onset, frequency/type of seizure, MRI findings, video-EEG, histopathology and Engel's grading at 1 year/last follow up were recorded. RESULTS: The duration of epilepsy at surgery was 9.9±6.9 years. The location of lesions were: temporal: 41 (60.3%); frontal: 21 (30.9%); parietal: 6 (8.8%). The type of lesionectomies performed were temporal 41 (60.3%), extra-temporal: 25 (36.8%), temporo-frontal and temporo-parietal: 1 (1.5%) patient each. The histopathological diagnosis were neoplastic: 32 (47.1%), cortical dysplasia: 19 (27.9%), other focal lesions: 17 (25%). At mean follow up of 2.9±2.1 years (median: 2.6 years), outcome was - Engel's class I: 43 (63.2%), IIa: 14 (20.6%), III: 7 (10.3%), IV: 4 (5.9%). Good seizure control (Engel's class I/IIa) was achieved in 57 (83.8%) patients. The good prognostic markers included temporal seizures, extended lesionectomy and AEDs after surgery while poor prognostic marker was gliotic lesion on histopathology. CONCLUSION: Following lesionectomy due to non-MTS lesions, seizure freedom (Engel I) was noted in about 63.2% of patients, which is comparable to other series and reiterates the effectiveness of lesionectomy for seizure control.

Sequence insertions in the HIV type 1 subtype C viral promoter predominantly generate an additional NF-κB binding site.

After screening a large number of clinical samples of HIV-1 subtype C in India, a subset of viral strains containing sequence insertions upstream of the viral enhancer has been identified. The sequence insertions contained binding sites for at least two different transcription factors NF-κB and RBEIII, importantly, in a mutually exclusive fashion. Furthermore, while some of the viral strains contained insertions of κB-like sites, a few others contained dual insertions of the RBEIII and κB sites together but only one of the two was intact. NF-κB acquisition appears to be the most common phenotype unique for subtype C with nearly half of the variant strains containing such insertions. Given that subtype C already contains three functional NF-κB sites in the viral enhancer, acquisition of a fourth NF-κB motif in some variant viral strains is intriguing. Further investigation is warranted to examine the significance of the sequence insertions for the replicative fitness of the variant viral strains.

Fatal status epilepticus: a clinico-pathological analysis among 100 patients: from a developing country perspective.

PURPOSE: We evaluated the neuropathological features associated with fatal SE with emphasis on neuroanatomy and etiology, and attempted to correlate with the clinical observations. MATERIALS AND METHODS: Details of 100 (n=100; M:F=64:36) autopsied cases of status epilepticus were studied retrospectively with emphasis on clinico-pathological characteristics. RESULTS: Sixty-five percent of patients presented for the first time as SE. Patients with prior history of epilepsy (35%), had discontinued treatment in 71.4% of cases, resulting in SE. Majority of patients with SE had generalized convulsive seizures (75%). The mean GCS score at admission was 4.4. Median delay in initiating the treatment following seizure was 23.5h (mean: 29.5±32.8h). The etiological factors could be identified in two-thirds of cases, neuroinfection (n=34) and stroke (n=16) being the commonest. Frontal lobe was the most commonly involved area (38.6%), in both localized and multilobar pathology. Dual pathology was observed in seven patients. Clinico-pathological concordance was noted in 58%. The discordance observed in 42 patients was either due to low index of suspicion for additional etiologies, inaccurate neuroimaging interpretation or non-availability of extensive diagnostic modalities prior to referring to this centre. CONCLUSIONS: This is one of the largest cohort of fatal SE with pathological correlation in the literature. Clinico-pathological concordance was observed in 58% of patients. Neuroinfection and cerebral stroke were observed in two-thirds of patients as the etiological factor. Prolonged duration of SE, long delay in initiating treatment, poor GCS score at admission, poor drug compliance and symptomatic etiologies were common in this cohort of fatal SE from a developing country, most of which could have been preventable.

Elemental mercury poisoning probably causes cortical myoclonus.

Mercury toxicity causes postural tremors, commonly referred to as "mercurial tremors," and cerebellar dysfunction. A 23-year woman, 2 years after injecting herself with elemental mercury developed disabling generalized myoclonus and ataxia. Electrophysiological studies confirmed the myoclonus was probably of cortical origin. Her deficits progressed over 2 years and improved after subcutaneous mercury deposits at the injection site were surgically cleared. Myoclonus of cortical origin has never been described in mercury poisoning. It is important to ask patients presenting with jerks about exposure to elemental mercury even if they have a progressive illness, as it is a potentially reversible condition as in our patient.

Melanized nigral neuronal numbers in Nigerian and British individuals.

The role of genetic and environmental factors in etiopathogenesis of Parkinson's disease (PD) is debated. The prevalence of PD is higher among white than nonwhite populations, yet it is five times higher in nonwhites living in the United States than in Nigeria. We compare counts of melanized nigral neurons between neurologically normal Nigerians and British brains. Neuronal counts were estimated in an age-matched sample of 23 Nigerian and 7 British brains from neurologically normal individuals who had no Lewy bodies and Lewy neurites on alpha-synuclein immunostaining. Two investigators blind to age and ethnicity performed counts of melanized neurons in a single 7-mum hemisections showing the substantia nigra pars compacta. No significant difference exits in the number of neurons between the Nigerian and the British subjects (P = 0.1, NS). Differences in melanized nigral neuronal numbers may not explain differences in the prevalence of PD between white and nonwhite populations, suggesting factors other than neuronal numbers contribute to differential susceptibility of black vs. white races to PD.