Fridoon J. Ahmad

Motor proteins regulate force interactions between microtubules and microfilaments in the axon

It has long been known that microtubule depletion causes axons to retract in a microfilament-dependent manner, although it was not known whether these effects are the result of motor-generated forces on these cytoskeletal elements. Here we show that inhibition of the motor activity of cytoplasmic dynein causes the axon to retract in the presence of microtubules. This response is obliterated if microfilaments are depleted or if myosin motors are inhibited. We conclude that axonal retraction results from myosin-mediated forces on the microfilament array, and that these forces are counterbalanced or attenuated by dynein-mediated forces between the microfilament and microtubule arrays.

Force generation by cytoskeletal motor proteins as a regulator of axonal elongation and retraction

Axons elongate and retract in response to environmental signals during the development of the nervous system. There is broad agreement that these signals must affect the cytoskeleton to elicit bouts of elongation or retraction. Most contemporary studies have speculated that bouts of elongation involve polymerization of the cytoskeleton whereas bouts of retraction involve depolymerization of the cytoskeleton. Here we present an alternative view, namely that molecular motor proteins generate forces on the cytoskeletal polymers that can affect their distribution and configuration. In this view, bouts of axonal elongation involve net forward movement of cytoskeletal elements whereas bouts of retraction involve net backward movements. We propose that environmental cues elicit bouts of elongation or retraction via biochemical pathways that modulate the activities of relevant motors.

Inhibition of microtubule nucleation at the neuronal centrosome compromises axon growth

We tested the dependence of axon growth on microtubule (MT) nucleation from the neuronal centrosome. Nocodazole diminished MTs in freshly plated neurons by > 99%. Within 5 min of drug removal, MTs reassembled at the centrosome. This response was inhibited in cells microinjected with gamma-tubulin antibody. Within 2 hr of drug removal, uninjected neurons grew > 500 microns of axon. In roughly half of the antibody-injected cells, axon growth was abolished and MT levels were reduced by approximately 87% compared with uninjected cells. In the other antibody-injected cells, axon growth was compromised but not abolished, and MT levels were reduced by approximately 38%. Thus inhibition of MT nucleation at the centrosome hindered MT reassembly, and depending on the severity of this response, axon growth was either compromised or abolished.

Effects of dynactin disruption and dynein depletion on axonal microtubules.

We investigated potential roles of cytoplasmic dynein in organizing axonal microtubules either by depleting dynein heavy chain from cultured neurons or by experimentally disrupting dynactin. The former was accomplished by siRNA while the latter was accomplished by overexpressing P50‐dynamitin. Both methods resulted in a persistent reduction in the frequency of transport of short microtubules. To determine if the long microtubules in the axon also undergo dynein‐dependent transport, we ascertained the rates of EGFP‐EB3 “comets” observed at the tips of microtubules during assembly. The rates of the comets, in theory, should reflect a combination of the assembly rate and any potential transport of the microtubule. Comets were intitally slowed during P50‐dynamitin overexpression, but this effect did not persist beyond the first day and was never observed in dynein‐depleted axons. In fact, the rates of the comets were slightly faster in dynein‐depleted axons. We conclude that the transient effect of P50‐dynamitin overexpression reflects a reduction in microtubule polymerization rates. Interestingly, after prolonged dynein depletion, the long microtubules were noticeably misaligned in the distal regions of axons and failed to enter the filopodia of growth cones. These results suggest that the forces generated by cytoplasmic dynein do not transport long microtubules, but may serve to align them with one another and also permit them to invade filopodia.

Beyond taxol: microtubule-based treatment of disease and injury of the nervous system

Contemporary research has revealed a great deal of information on the behaviours of microtubules that underlie critical events in the lives of neurons. Microtubules in the neuron undergo dynamic assembly and disassembly, bundling and splaying, severing, and rapid transport as well as integration with other cytoskeletal elements such as actin filaments. These various behaviours are regulated by signalling pathways that affect microtubule-related proteins such as molecular motor proteins and microtubule severing enzymes, as well as a variety of proteins that promote the assembly, stabilization and bundling of microtubules. In recent years, translational neuroscientists have earmarked microtubules as a promising target for therapy of injury and disease of the nervous system. Proof-of-principle has come mainly from studies using taxol and related drugs to pharmacologically stabilize microtubules in animal models of nerve injury and disease. However, concerns persist that the negative consequences of abnormal microtubule stabilization may outweigh the positive effects. Other potential approaches include microtubule-active drugs with somewhat different properties, but also expanding the therapeutic toolkit to include intervention at the level of microtubule regulatory proteins.

Association of generalized vitiligo with MHC class II loci in patients from the Indian subcontinent.

TO THE EDITOR Generalized vitiligo is a disease in which patches of depigmented skin and overlying hair result from autoimmune destruction of melanocytes in involved regions (Spritz, 2012). Clinic-based studies cite high prevalence of vitiligo in India, up to 8.8% (e.g. Handa and Kaur, 1999), though population-based surveys report much lower prevalence, 0.46% in Calcutta (Das et al., 1985) and 1.79% in South Gujarat (Mehta et al., 1973). Vitiligo is a distressing cosmetic problem in individuals of dark skin phototypes, due to striking contrast between lesions and unaffected skin. This may explain the reported high prevalence of vitiligo in India and negative impact on perceived quality of life in this population (Parsad et al., 2003). Indeed, vitiligo has long been recognized in India (Singh et al., 1974), the specific use of ultraviolet light treatment was pioneered in India (Menon, 1945), and some of the earliest genetic studies of vitiligo were carried out there: of ABO blood groups, α1-antitrypsin, and haptoglobin, and subsequent candidate gene studies, including GCH1, ACE, CAT, CTLA4, GPX1, IL4, MBL2, and PTPN22, most yielding negative or conflicting results. Recently, Singh et al. (2012) tested genetic association of vitiligo in Indian patients with HLA–A, -B, -C in the MHC class I region and HLA-DRB1 in the class II region, identifying primary genetic association with HLA-DRB1* 07:01. Here, we describe a more comprehensive genetic association study of generalized vitiligo on the Indian subcontinent, utilizing the Immunochip® (Cortes and Brown, 2011) to screen 196,524 SNPs in 128 loci previously implicated in autoimmune and inflammatory diseases, including 9441 SNPs spanning the extended major histocompatibility complex (MHC) on chromosome 6p. Our results suggest there are at least two independent association signals in the MHC class II region, one located upstream of HLA-DRA and the other located between HLA-DRB1 and HLA-DQA1, generally similar to what we previously found in a genomewide association study of vitiligo in European-derived whites (EUR) (Jin et al., 2010). Our initial study group consisted of 255 patients with generalized vitiligo and 377 unrelated non-vitiligo controls of Indian subcontinent (Pakistan, India, Sri Lanka, Bangladesh) derivation. After quality control procedures, data for 120,724 remaining SNPs from 251 remaining cases were compared to those from 349 remaining controls. Suggestive association signals were considered as clusters of nearby SNPs with trend P-values <10−5. The International Immunochip Consortium has agreed on a genomewide significance criterion of P<5 × 10−8 for studies utilizing the Immunochip (Cortes and Brown, 2011). As shown in Figure 1a and Supplementary Table S1, the only highly suggestive association signals were in the MHC class II gene region (Figure 1b), from rs3134942 (chr6:32168770) to rs2856674 (chr6:32659644), spanning the upstream part of NOTCH4 through HLA-DQB1. The principal region of association encompassed c6orf10--BTNL2--HLA-DRA--HLA-DRB5--HLA-DRB1--HLA-DQA1 (Figure 1b), with extensive LD through this region in this population (Figure 1c). One SNP, rs482044, located towards the centromeric end of the region, between HLA-DRB1 and HLA-DQA1, achieved genomewide significance (G allele; P=1.94 × 10−8, OR=1.93; Table 1), remaining significant (P = 4.86 × 10−8) even after correction for the observed genomic inflation factor λ = 1.06. Figure 1 Immunochip association results for generalized vitiligo Table 1 MHC class II region SNPs genotyped in Immunochip screening and in replication studies To determine which SNPs in the MHC class II region represent primary association with vitiligo versus are signals secondary to LD, we applied a backward regression procedure, comparing a model including the seven most significant MHC class II SNPs to alternative models in which each SNP was removed one by one. This analysis suggested that this region contains two independent associated loci, one represented by rs482044-G (located between HLA-DRB1 and HLA-DQA1) and the other represented by rs3129859-C (located 6680 nt upstream of HLA-DRA). Forward regression analysis of these two SNPs showed that the model composed of rs3129859 was significantly (P=4.4 × 10−5) improved by adding rs482044, and that the model composed of rs482044 was significantly improved (P=6.0 × 10−4) by adding rs3129859. In contrast to our previous findings in EUR (Jin et al., 2010), we observed no apparent association of vitiligo with SNPs in the MHC class I region in this Indian-Pakistani population (Figures 1a and 1b). Furthermore, considering loci represented on the Immunochip that have been reported to be associated with vitiligo in previous candidate gene studies from India, no SNPs in the ACE (3 SNPs), CTLA4 (505 SNPs), or IL4 (103 SNPs) gene regions showed even nominal association in the present study. To confirm association of generalized vitiligo with MHC class II region SNPs in the Indian subcontinent, we carried out a replication study of rs3129859 and rs482044, as well as the third most significant Immunochip SNP, rs3096691 (located just upstream of NOTCH4) (Fig. 1b). These three SNPs were genotyped in 685 unrelated generalized vitiligo cases and 774 unrelated controls from Gujarat state, India. All three were in Hardy-Weinberg equilibrium in the controls, and all three achieved at least nominal significance in the replication study (Table 1). Most significant association in the replication study was observed for rs3129859-C (P=9.48 × 10−9), with no significant heterogeneity of OR between the two studies (PBreslow-Day=1.15 × 10−1). Cochran-Mantel-Haenszel meta-analysis of the rs3129859 data from the Immunochip screen and replication study likewise yielded strongest overall association (P=4.30 × 10−14, OR=1.67; 95% C.I. 1.46–1.91). Association was also confirmed in the replication study for rs482044 (P= 1.11 × 10−4), with only nominal association for rs3096691 (P=2.32 × 10−2), although both of these SNPs exhibited heterogeneity of OR. Both rs482044 (P=1.58 × 10−2) and rs3129859 (P = 1.20 × 10−6) remained significant when each was conditioned on the other. Overall, our findings thus generally confirm association of vitiligo with at least two independent loci in the MHC class II region. In a previous genomewide-association study of generalized vitiligo in EUR subjects, we found that both vitiligo susceptibility (Jin et al., 2010) and age of onset (Jin et al., 2011) are likewise associated with at least two independent loci in the MHC class II region. To assess whether the MHC class II loci observed in the Indian subcontinent and EUR populations might correspond ancestrally, we carried out trans-ethnic meta-analysis using MANTRA (Morris, 2011), which indicated that the MHC association signal represented by rs482044 in the Indian subcontinent population apparently corresponds to the MHC signal represented by rs532098 in EUR (Jin et al., 2010) (Table S2). In contrast, rs3129859 is not significantly associated with vitiligo in EUR (Jin et al., 2010), and correspondence between the association signals upstream of HLA-DRA observed in both populations remains uncertain. Our findings thus highlight both similarity and differences of vitiligo MHC genetic associations in subjects from different major world populations. On the Indian subcontinent, this study and that of Singh et al. (2012) support association of vitiligo with loci in the MHC class II region, but show no primary association in the MHC class I region. Similarly, in the EUR population, vitiligo is also associated with multiple signals in the MHC class II region, at least one of which, between HLA-DRB1 and HLA-DQA1, appear to correspond to one in the Indian subcontinent population. However, in the EUR population vitiligo shows primary association with HLA-A in the distal class I region (Jin et al., 2010); specifically, HLA-A*02:01 (Jin et al., 2011). In addition, studies in Chinese show principal MHC association in the class III region (Quan et al., 2010) and in the proximal class I region, between HLA-B and HLA-C (Liu et al., 2012). Together, these similarities and differences of principal MHC genetic associations with generalized vitiligo among different populations may in part underlie differing prevalence of this autoimmune disease in different groups around the world.

Cytoplasmic abnormalities in cultured cerebellar neurons from the trisomy 16 mouse

This study represents a first effort to characterize the growth and development of murine trisomy 16 neurons using single-cell neuron culture techniques. Murine trisomy 16 is a model for the human Down syndrome, or trisomy 21. Both show similar nervous system abnormalities including decreases in cerebellar size and in numbers of cerebellar neurons. Trisomy 16 cerebellar neurons cultured from 17-gestational day conceptuses grew less extensive neuritic arbors than normal neurons. Unlike controls, the individual neurites of the trisomic neurons were not clearly distinguishable as axons or dendrites over the 10 day period that they were observed. The trisomic neurons were characterized by diminished levels of microtubules, abnormally shaped mitochondria, and the presence of dense bundles of abnormal filaments that were not observed in any of the normal littermate neurons.

Towards Better Understanding of the Pathogenesis of Neuronal Respiratory Network in Sudden Perinatal Death

Sudden perinatal death that includes the victims of sudden infant death syndrome, sudden intrauterine death syndrome, and stillbirth are heartbreaking events in the life of parents. Most of the studies about sudden perinatal death were reported from Italy, highlighting two main etiological factors: prone sleeping position and smoking. Other probable contributory factors are prematurity, male gender, lack of breastfeeding, respiratory tract infections, use of pacifiers, infant botulism, extensive use of pesticides and insecticides, etc. However, extensive studies across the world are required to establish the role of these factors in a different subset of populations. Previous studies confirmed the widely accepted hypothesis that neuropathology of the brainstem is one of the main cause of sudden perinatal death. This study is an effort to summarize the neuropathological evaluation of the brainstems and their association to sudden perinatal death. Brainstem nuclei in vulnerable infants undergo certain changes that may alter the sleep arousal cycle, cardiorespiratory control, and ultimately culminate in death. This review focuses on the roles of different brainstem nuclei, their pathologies, and the established facts in this regard in terms of it’s link to such deaths. This study will also help to understand the role of brainstem nuclei in controlling the cardiorespiratory cycles in sudden perinatal death and may provide a better understanding to resolve the mystery of these deaths in future. It is also found that a global initiative to deal with perinatal death is required to facilitate the diagnosis and prevention in developed and as well as developing countries.

Role of neurotransmitter Substance P in progression of oral squamous cell carcinoma

BACKGROUND Oral squamous cell carcinoma (OSCC) is the most frequent type of head and neck cancers. OBJECTIVES In the present study, we evaluated the expression and distribution of Substance P (SP) in different grades of OSCC and role of SP in its proliferation and progression. SUBJECTS AND METHODS Forty OSCC biopsies were immunohistochemically analyzed by using SP antibody, including 29 male and 11 female cases. 35% were well differentiated, 35% moderately differentiated and 30% poorly differentiated OSCC. The majority of patients were in the age range of 41-80 years. 62% of the cases were positive for SP. SP positivity was expressed in the cytoplasm of the tumor cells. Most of the positive cases were from the tongue region. RESULTS 93% of moderately differentiated, 92% of poorly differentiated and 8% of well-differentiated carcinomas were SP-positive, but SP expression intensity was highest in poorly differentiated cases (+3). More positive patients were males (68.96% of all male patients) with moderately and poorly differentiated OSCC. Among all positive cases, 48% were poorly differentiated, 48% moderately differentiated and 4% well differentiated. CONCLUSION Strong expression of SP in poorly and moderately differentiated cases suggests a role of SP in the progression and development of tumor. Expression of SP in the current study increased as the proliferation of cells increased. Prevalence of oral cancer in males may be due to the fact that they smoke and use pan, chewing gum, beetle nut etc. in this region. SP antagonists can help in the reduction and inhibition of oral cancer. SP has a diagnostic value with sensitivity of 92.5% and specificity of 93.7%. The positive predictive value is 96.2% and the negative predictive value 88.2%.

Genetic analysis of prolactin gene in Pakistani cattle

Prolactin (PRL) is a polypeptide hormone, secreted mainly by the anterior pituitary gland. It is involved in many endocrine activities. The key functions of PRL are related to reproduction and lactation in mammals. To ascertain the presence of polymorphisms in the bovine PRL gene (bPRL), the bPRL gene was sequenced. Five mutations were identified in exonic region and eleven in associated intronic regions in 100 cattle from four Pakistani cattle breeds. Haplotype of predicted amino acid changes represent a common alteration at codon 222 from R-Arginine into K-Lysine in all four breeds. Significant statistical variations were observed in the distribution of single nucleotide polymorphism (SNP) in various cattle populations. However, on basis of present study, an association of these SNPs with milk performance traits in four Pakistani cow breeds cannot be truly replicated but at least can be effective DNA markers for some of the breeds studied. Linkage analysis between these SNPs on larger populations can be useful for the association with milk production traits. Furthermore, present study may be used for marker-assisted selection and management in cattle breeding program in local cattle breeds.