Control Brain Extracts Research Articles

Pathological changes induced by Alzheimer’s brain inoculation in amyloid-beta plaque-bearing mice

Alzheimer's disease (AD) is characterized by intracerebral accumulations of extracellular amyloid-β (Aβ) plaques and intracellular tau pathology that spread in the brain. Three types of tau lesions occur in the form of neuropil threads, neurofibrillary tangles, and neuritic plaques i.e. tau aggregates within neurites surrounding Aβ deposits. The cascade of events linking these lesions and synaptic or memory impairments are still debated. Intracerebral infusion of human AD brain extracts in Aβ plaque-bearing mice that do not overexpress pathological tau proteins induces tau pathologies following heterotopic seeding of mouse tau protein. There is however little information regarding the downstream events including synaptic or cognitive repercussions of tau pathology induction in these models. In the present study, human AD brain extracts (ADbe) and control-brain extracts (Ctrlbe) were infused into the hippocampus of Aβ plaque-bearing APPswe/PS1dE9 mice. Memory, synaptic density, as well as Aβ plaque and tau aggregate loads, microgliosis, astrogliosis at the inoculation site and in connected regions (perirhinal/entorhinal cortex) were evaluated 4 and 8 months post-inoculation. ADbe inoculation produced the following effects: (i) memory deficit; (ii) increased Aβ plaque deposition in proximity to the inoculation site; (iii) tau pathology induction; (iv) appearance of neuropil threads and neurofibrillary tangles next to the inoculation site with a spreading to connected regions. Neuritic plaque pathology was detected in both ADbe- and Ctrlbe-inoculated animals but ADbe inoculation increased the severity close to and at distance of the inoculation site. (v) Finally, ADbe inoculation reduced synaptic density in the vicinity to the inoculation site and in connected regions as the perirhinal/entorhinal cortex. Synaptic impairments were correlated with increased severity of neuritic plaques but not to other tau lesions or Aβ lesions, suggesting that neuritic plaques are a culprit for synaptic loss. Synaptic density was also associated with microglial load.Graphical abstract

Transmission of amyloid-beta and tau pathologies is associated with cognitive impairments in a primate

Amyloid-β (Aβ) pathology transmission has been described in patients following iatrogenic exposure to compounds contaminated with Aβ proteins. It can induce cerebral Aβ angiopathy resulting in brain hemorrhages and devastating clinical impacts. Iatrogenic transmission of tau pathology is also suspected but not experimentally proven. In both scenarios, lesions were detected several decades after the putatively triggering medico-surgical act. There is however little information regarding the cognitive repercussions in individuals who do not develop cerebral hemorrhages. In the current study, we inoculated the posterior cingulate cortex and underlying corpus callosum of young adult primates (Microcebus murinus) with either Alzheimer’s disease or control brain extracts. This led to widespread Aβ and tau pathologies in all of the Alzheimer-inoculated animals following a 21-month-long incubation period (n = 12) whereas none of the control brain extract-inoculated animals developed such lesions (n = 6). Aβ deposition affected almost all cortical regions. Tau pathology was also detected in Aβ-deposit-free regions distant from the inoculation sites (e.g. in the entorhinal cortex), while some regions adjacent, but not connected, to the inoculation sites were spared (e.g. the occipital cortex). Alzheimer-inoculated animals developed cognitive deficits and cerebral atrophy compared to controls. These pathologies were induced using two different batches of Alzheimer brain extracts. This is the first experimental demonstration that tau can be transmitted by human brain extracts inoculations in a primate. We also showed for the first time that the transmission of widespread Aβ and tau pathologies can be associated with cognitive decline. Our results thus reinforce the need to organize a systematic monitoring of individuals who underwent procedures associated with a risk of Aβ and tau iatrogenic transmission. They also provide support for Alzheimer brain-inoculated primates as relevant models of Alzheimer pathology.

N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain

Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions.Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau.Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.

Abstract 4296: Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells

Abstract Background: Although interfering with the actions of microtubule-associated proteins (MAP’s) is successful against ovarian epithelial carcinoma (OVCA), knowledge of the MAP’s in OVCA cells is lacking. We sought to identify cancer-specific MAP’s in OVCA cells.Methods: Four human OVCA cell lines, primary OVCA tissue, ascitic cells and metastases from 9 patients and normal ovarian tissue from six women were compared to control human and rat brain tissue, uterine myomas (benign tumors) and rat and human non-neural organs. Modified Western blotting using panels of anti-microtubule-associated protein monoclonal antibodies were used. An in-vitro brain and OVCA mixing and recovery experiment was carried out to assess possible degradation of MAP1a by OVCA. A paclitaxel-driven tubulin assembly assay was performed to determine whether the OVCA protein has microtubule-associated protein properties. The transcriptome of CSP-80 was sequenced using primer extension sequencing. Results: (1) In contradistinction to the 350 kDa MAP1a present in control brain extracts, only an 80 kDa molecule with MAP1a immune-reactivity was found in OVCA cell lines and tissues. No immune-reactivity was present in the negative controls. (2)Following the mixing experiments there was no increase of CSP-80; CSP-80 is not a degradation artifact of MAP1a. (3) The paclitaxel-driven tubulin assembly assay showed that CSP-80 bound to microtubules during tubulin polymerization. (4) No CSP-80 was identified in human or rat non-malignant tissues including normal human and rat brain or normal human ovary, placenta or uterine myomas, or normal rat liver, spleen or lung. (5) cDNA sequencing shows a 2603 bp fragment at a 5’ terminal of p80 to be homologous with the analogous segment of MAP1a. (6) Further testing showed that CSP-80 was present in other reproductive and non-reproductive carcinomas. Again, no 350 kDa MAP1a was present. Studies are underway to determine whether CSP-80 is a mutated protein or the product of post-transcriptional modification. Conclusion: CSP-80 is a novel cancer-related protein that appears to be limited to an 80 kDa sequence of the MAP1a gene, or a post-transcriptionally modified product. Since CSP-80 exhibits tubulin-binding activity, this may explain the efficacy of taxane derivatives. CSP-80 may disclose some aspects of carcinogenesis, be a valuable marker of cancer and therapeutic efficacy, and offer new avenues for cancer treatment. Citation Format: Ahmed Fadiel, Viswam S. Nair, Peter Aceto, Frederick Naftolin. Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4296.

Deep Sequencing for the Detection of Virus-Like Sequences in the Brains of Patients with Multiple Sclerosis: Detection of GBV-C in Human Brain

Multiple sclerosis (MS) is a demyelinating disease of unknown origin that affects the central nervous system of an estimated 400,000 Americans. GBV-C or hepatitis G is a flavivirus that is found in the serum of 1–2% of blood donors. It was originally associated with hepatitis, but is now believed to be a relatively non-pathogenic lymphotropic virus. Fifty frozen specimens from the brains of deceased persons affected by MS were obtained along with 15 normal control brain specimens. RNA was extracted and ribosomal RNAs were depleted before sequencing on the Illumina GAII. These 36 bp reads were compared with a non-redundant database derived from the 600,000+ viral sequences in GenBank organized into 4080 taxa. An individual read successfully aligned to the viral database was considered to be a “hit”. Normalized MS specimen hit rates for each viral taxon were compared to the distribution of hits in the normal controls. Seventeen MS and 11 control brain extracts were sequenced, yielding 4–10 million sequences (“reads”) each. Over-representation of sequence from at least one of 12 viral taxa was observed in 7 of the 17 MS samples. Sequences resembling other viruses previously implicated in the pathogenesis of MS were not significantly enriched in any of the diseased brain specimens. Sequences from GB virus C (GBV-C), a flavivirus not previously isolated from brain, were enriched in one of the MS samples. GBV-C in this brain specimen was confirmed by specific amplification in this single MS brain specimen, but not in the 30 other MS brain samples available. The entire 9.4 kb sequence of this GBV-C isolate is reported here. This study shows the feasibility of deep sequencing for the detection of occult viral infections in the brains of deceased persons with MS. The first isolation of GBV-C from human brain is reported here.

Truncation and Activation of Calcineurin A by Calpain I in Alzheimer Disease Brain

A disturbance of calcium homeostasis is believed to play an important role in the neurodegeneration of the brains of Alzheimer disease (AD) patients, but the molecular pathways by which it contributes to the disease are not well understood. Here we studied the activation of two major Ca(2+)-regulated brain proteins, calpain and calcineurin, in AD brain. We found that calpain I is activated, which in turn cleaves and activates calcineurin in AD brain. Mass spectrometric analysis indicated that the cleavage of calcineurin by calpain I is at lysine 501, a position C-terminal to the autoinhibitory domain, which produces a 57-kDa truncated form. The 57-kDa calcineurin maintains its Ca(2+)/calmodulin dependence of the phosphatase activity, but the phosphatase activity is remarkably activated upon truncation. The cleavage and activation of calcineurin correlate to the number of neurofibrillary tangles in human brains. These findings suggest that the overactivation of calpain I and calcineurin may mediate the role of calcium homeostatic disturbance in the neurodegeneration of AD.

Huntingtin is degraded to small fragments by calpain after ischemic injury☆

The gene defect in Huntington’s disease (HD) causes a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). In vitro studies suggest that mutant N-htt fragments can aggregate and cause cell death in HD. The physiological and pathological conditions that affect htt proteolysis in the brain are unclear. We examined htt expression by Western blot in the rat brain after transient ischemic injury, which causes striatal neurodegeneration similar to that seen in HD and activates proteases including calcium-dependent calpains. Focal brain ischemia reduced levels of full-length htt in the infarcted cortex and striatum and increased expression of a 55-kDa N-htt fragment that was also produced by treating control brain extracts with calpain. N-htt fragments between 65 and 80 kDa also rose after injury, but these fragments were not as long-lived as the 55-kDa N-htt fragment. The results suggest that after ischemic injury full-length htt is degraded in degenerating neurons and an N-htt fragment accumulates.

Topographic regulation of kinase activity in Alzheimer's disease brains.

At autopsy, a most distinctive pathology seen in Alzheimer's disease (AD) brains is numerous abnormal neurons filled with neurofibrillary tangles (NFTs) containing stable complexes of hyperphosphorylated tau (PHF), neurofilaments and various kinases, among other proteins. Though these neuronal aggregates have been actively studied, their nature and origin are still poorly understood. Our studies of regulation of phosphorylation in neurons of the squid giant fiber system, using P13(suc1) affinity chromatography, suggest that neuronal phosphorylation of cytoskeletal proteins is compartmentalized into active axonal and inactive cell body-specific multimeric complexes of kinases, substrates and phosphatases. To determine whether such compartment-specific phosphorylation complexes are present in human brains, we separated gray matter (enriched in cell bodies) and white matter (enriched in axons) from normal and AD brains and studied the total kinase activities in lysates, pellets and P13(suc1) complexes. In addition, Western blot analysis was used to characterize the proteins associated with P13(suc1) multimeric complexes extracted from gray and white matter. We tested the hypothesis that P13 phosphorylation complexes were abnormally compartmentalized in AD neurons with the more active complexes shifted to cell bodies (gray matter) instead of axons (white matter). We found that (1) endogenous and exogenous substrate-dependent kinase activities of AD and control brain extracts were similar in both gray and white matter. (2) Long post mortem times tend to erase any differences in kinase activity between control and AD extracts. In contrast to shorter post mortem times (4.5-10 hrs), long post mortem times (13-34 hrs) significantly minimize the variances in kinase activities between control and AD brain extracts suggesting that cell death and proteolysis may eliminate any intrinsic differences in enzyme activities. (3) Except for the significantly higher level of histone phosphorylation in control white extracts, the kinase activities of P13(suc1)-derived multimeric complexes from gray and white matter were also similar in control and AD brains. Here, too, variances between control and AD distributions were significantly different (p < 0.001-0.02) suggesting that the P13 complexes were different. We also found differences in the Western blot profiles of P13suc1-associated kinases and cytoskeletal proteins; higher expression of phosphorylated NF-H and PHF-tau in gray matter of AD brains was detected. We believe that such differences in P13 complexes from human control and AD brain samples displaying extensive heterogeneity in age, post mortem time and clinical history, may be important.

Protein oxidation in the brain in Alzheimer's disease

In this study we used immunohistochemistry and two-dimensional fingerprinting of oxidatively modified proteins (two-dimensional Oxyblot) together to investigate protein carbonyl formation in the Alzheimer's disease brain. Increased protein oxidation was detected in sections from the hippocampus and parahippocampal gyrus, superior and middle temporal gyri of six Alzheimer's disease and six age-matched control human subjects, but not in the cerebellum. In two brain regions severely affected by Alzheimer's disease pathology, prominent protein carbonyl immunoreactivity was localized in the cytoplasm of neurons without visual pathomorphological changes and degenerating neurons, suggesting that intracellular proteins might be significantly affected by oxidative modifications. Following two-dimensional electrophoresis the positions of some individual proteins were identified using specific antibodies, and immunoblot analysis for protein carbonyls was performed. These studies demonstrated the presence of protein carbonyl immunoreactivity in β-tubulin, β-actin and creatine kinase BB in Alzheimer's disease and control brain extracts. Protein carbonyls were undetectable in spots matching glial fibrillary acidic protein and tau isoforms. Specific protein carbonyl levels in β-actin and creatine kinase BB were significantly higher in Alzheimer's disease than in control brain extract. β-Tubulin did not demonstrate a significant increase in specific protein carbonyl content in Alzheimer's disease brains. We suggest that oxidative stress-induced injury may involve the selective modification of different intracellular proteins, including key enzymes and structural proteins, which precedes and may lead to the neurofibrillary degeneration of neurons in the Alzheimer's disease brain.

Oxidative modification of creatine kinase BB in Alzheimer's disease brain.

Creatine kinase (CK) BB, a member of the CK gene family, is a predominantly cytosolic CK isoform in the brain and plays a key role in regulation of the ATP level in neural cells. CK BB levels are reduced in brain regions affected by neurodegeneration in Alzheimer's disease (AD), Pick's disease, and Lewy body dementia, and this reduction is not a result of decreased mRNA levels. This study demonstrates that posttranslational modification of CK BB plays a role in the decrease of CK activity in AD brain. The specific CK BB activity and protein carbonyl content were determined in brain extracts of six AD and six age-matched control subjects. CK BB activity per microgram of immunoreactive CK BB protein was lower in AD than in control brain extracts, indicating the presence of inactive CK BB molecules. The analysis of specific protein carbonyl levels in CK BB, performed by two-dimensional fingerprinting of oxidatively modified proteins, identified CK BB as one of the targets of protein oxidation in the AD brain. The increase of protein carbonyl content in CK BB provides evidence that oxidative posttranslational modification of CK BB plays a role in the loss of CK BB activity in AD.

Phosphorylation Activity in the Alzheimer's Disease and Normal Brain is Modulated by Microtubule-Associated Protein, Tau in Vitro.

One of the hallmarks of Alzheimer's Disease is the presence of abundant neurofibrillary tangles (NFTs) in the brains of affected individuals. Hyperphosphorylated tau is a major component of paired helical filaments (PHFs) in NFTs. Tau is a neuronal microtubule associated protein found primarily in axons. Normal tau promotes tubulin polymerization and stabilizes microtubule (MT) structures, whereas hyperphosphorylated tau reduces its affinity for MTs and destabilizes MT-structures. This results in the disruption of vital cellular processes (e.g. axonal transport) and leads to the degeneration of affected neurons. Processes leading to the hyperphosphorylation of tau and formation of neurofibrillary lesions in Alzheimer's Disease (AD) brains are not understood. Phosphorylation of a substrate molecule like tau depends upon the equilibrium between kinase and phosphatase activities and the availability of their substrate molecules in a given system. Therefore, to understand the relative roles of kinase and phosphatase activities, we studied the long-term kinetics of phosphorylation in AD and control brain extracts in the presence and absence of the phosphatase inhibitor okadaic acid (OA) using histone, casein and bacterially expressed tau as exogenous substrates. It was found that both kinase and phosphatase activities were higher in AD compared to control brains. Surprisingly, between 18 and 24 hours, there was a robust increase in phosphorylation of endogenous proteins in the brain extracts only when bacterially expressed tau was present in the phosphorylation reaction mixture. This pattern of phosphorylation activity was unaffected by OA. Significant difference in the phosphorylation of tau isoforms was also seen during this period. These data suggest that the expression and differential phosphorylation of certain tau isoforms may be responsible for the robust increase in phosphorylation and may play an important role in Alzheimer's pathology.

Estrogen increases the expression of uterine protein kinase C isozymes in a tissue specific manner

The pattern of protein kinase C isozyme expression in uterine smooth muscle and ventricular cardiac muscle was examined in ovariectomized rats pretreated with estradiol-17β alone or with estradiol-17β and progesterone. Protein kinase C isozyme expression was examined in membrane and cytosolic subcellular fractions by immunoblot analysis using antisera specific for α, γ, β1, β2, δ, ε, ζ, and θ isozymes. All isozymes were detectable in positive control brain extracts. The predominant isozymes in the myometrium were δ and β2 while in the ventricle, β2 and ζ were the dominant forms. In unstimulated tissues, all isozymes except PKC-δ, were predominantly found in the cytosolic compartment. Both estrogen and progesterone increased membrane-associated isozyme expression 35–125% in uterine muscle. Neither estrogen nor progesterone treatment significantly affected protein kinase C expression in cardiac muscle. These data suggest that estradiol, which increases uterine muscle hypertrophy and contractility, may exert these effects by increasing membrane-associated protein kinase C expression in a tissue-specific manner.

Hageman factor and its binding sites are present in senile plaques of Alzheimer's disease

Hageman factor (HF) or factor XII participates in several defense systems of the body. These include coagulation, fibrinolysis and complement activation. We investigated the expression of HF and its mRNA in control and Alzheimer's disease (AD) brain, using immunohistochemistry and polymerase-chain reaction (PCR) techniques. HF mRNA was detected in control and AD brain extracts, indicating that HF can be produced by endogenous brain cells. HF-like immunoreactivity was present in residual serum of capillaries in both control and AD brain, consistent with its known presence in the circulation. In addition, AD senile plaques were stained. The staining was dramatically enhanced when AD sections were incubated with solutions containing HF, indicating that plaques contain not only HF but also binding sites for HF. The enhanced staining was eliminated by pretreatment of solutions with the HF-binding agent kaolin. It was also eliminated by pretreatment of sections with protamine, an agent which strongly binds to negative surfaces. These data suggest that negatively charged surfaces in plaques might bind HF in vivo. Since HF can be activated by contact with negative surfaces, locally released HF could be playing a role in initiating a variety of inflammatory responses in AD brain.

Scrapie associated fibril detection on decomposed and fixed ovine brain material

Samples of cerebral cortex from eight scrapie affected sheep and two unaffected control sheep were stored for up to nine days at temperatures ranging from 18°C to 29°C. Scrapie associated fibrils ( saf) could be detected in proteinase K treated brain extracts from all the eight scrapie affected animals after rive days storage and in six out of the eight after nine days storage. SAF could not be detected in any brain extracts from the two control animals. Formol saline fixed brain material from a further six scrapie affected and two clinically normal sheep, were also subjected to an extraction technique used to detect fibrils. No characteristic saf were observed in any of these fixed samples. Long filamentous structures were observed in four of the fixed scrapie affected brain extracts and in one of the fixed unaffected control brain extracts.

Towards an improved survival of rat brain neurons in culture by cerebrospinal fluid of patients with senile dementia of Alzheimer's type

Neuronal cell survival was investigated in rat brain cortical cultures in the presence of increaseing concentrations of human brain extracts or cerebrospinal fluid (CSF) from control and Senile Dementia of Alzheimer's type (SDAT) patients. Using hippocampal brain extracts, converted 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was compared to the content of the neuronal marker MAP2 in foeral rat brain neuronal cultures in order to test converted MTT as a quantitative parameter for neuronal cell survival. A significant correlatio was found between both parameters. SDAT frontal cortex brain extracts induced a two to four-fold increase in neuronal cell survival at 25 to 125 μg protein extract, whereas control brain extracts induced at similar protein concentrations a decline in neuronal cell survival. The enhanced survival yielded by SDAT brain extracts was fully abolished in the presence of control brain extract. Control CSF concentration-dependently increased neuronal cell survival in postnatal rat brain neuronal cultures independent of the difference in the protein content of CSF samples and age of the patients. SDAT CSF also concentration-dependently enhanced neuronal cell survival, however, the effect was more pronounced compared to control CSF. These observations are in favour of the hypothesis that there might a higher neurotrophic activity in SDAT brain tissue.

Brain concentrations of benzodiazepines are elevated in an animal model of hepatic encephalopathy.

Brain extracts from rats with hepatic encephalopathy due to thioacetamide-induced fulminant hepatic failure contained 4- to 6-fold higher concentrations of substances that inhibit radioligand binding to benzodiazepine receptors than corresponding control rat extracts. Both isocratic and gradient-elution HPLC indicated that this inhibitory activity was localized in 3-8 peaks with retention times corresponding to deschlorodiazepam, deschlorolorazepam, lorazepam, oxazepam, diazepam, and N-desmethyldiazepam. The presence of diazepam and N-desmethyldiazepam was confirmed by mass spectroscopy. Both mass spectroscopic and radiometric techniques indicated that the concentrations of N-desmethyldiazepam and diazepam in brain extracts from encephalopathic rats were 2-9 and 5-7 times higher, respectively, than in control brain extracts. While benzodiazepines have been identified previously in mammalian and plant tissues, this report demonstrates that concentrations of these substances are increased in a pathophysiological condition. These findings provide a rational basis for the use of benzodiazepine receptor antagonists in the management of hepatic encephalopathy in humans.

Effects of azadirachtin on levels of ecdysteroids and prothoracicotropic hormone-like activity in Heliothis virescens (Fabr.) larvae

Oral injections of 1 μg of the plant chemical azadirachtin into final (fifth)-instar larvae of the tobacco budworm, Heliothis virescens (Fabr.), reduced whole body and haemolymph titres of the moulting hormones, ecdysone and 20-hydroxyecdysone, below those of control larvae, thereby preventing normal development to the pupal stage. Likewise, oral injections of 1 μg of azadirachtin reduced brain levels of prothoracicotropic hormone (PTTH)-like activity, which is the stimulation of in vitro moulting hormone synthesis by the prothoracic glands. In addition, prothoracic glands from azadirachtin-treated larvae were less receptive to stimulation in vitro and in vivo by control brain extracts of PTTH compared to control glands. However, azadirachtin did not directly inhibit the in vitro synthesis of moulting hormone when the compound was included in the incubation media with prothoracic glands from control larvae. A timely haemocoel injection of 20-hydroxyecdysone into azadirachtin-treated larvae resulted in partial recovery of development to the pupal stage. Based upon these data, we suggest that in preventing normal development of final-instar larvae of H. virescens, azadirachtin reduces moulting hormone titres by reducing PTTH titres and the receptivity of prothoracic glands to produce ecdysone via stimulation by PTTH.