Efficacy of dual intracerebroventricular and intravitreal CLN5 gene therapy in sheep prompts the first clinical trial to treat CLN5 Batten disease.

Mutations in the CLN5 gene cause the fatal, pediatric, neurodegenerative disease CLN5 neuronal ceroid lipofuscinosis. Affected children suffer progressive neuronal loss, visual failure and premature death. Presently there is no treatment. This study evaluated dual intracerebroventricular (ICV) and intravitreal (IVT) administration of a self-complementary adeno-associated viral vector encoding ovine CLN5 (scAAV9/oCLN5) into CLN5 affected sheep (CLN5-/-) at various disease stages. CLN5 disease progression was slowed in pre-symptomatic sheep who received a moderate dose of scAAV9/oCLN5, whilst a higher ICV dose treatment in early and advanced symptomatic animals delayed or halted disease progression. Intracranial (brain) volume loss was attenuated in all treatment cohorts, and visual function was also sustained in both the early and advanced symptomatic treated sheep over the 24-month duration of the study. Robust CLN5 protein expression was detected throughout the brain and spinal cord, and improvements in central nervous system and retinal disease correlates were observed. These findings hold translational promise for extending and improving the quality of life in both pre-symptomatic and symptomatic CLN5 patients, and prompted the initiation of the first in-human Phase I/II clinical trial testing ICV/IVT administration of scAAV9 encoding human CLN5 (https://clinicaltrials.gov/; NCT05228145).

Adolescent intermittent alcohol exposure produces strain-specific cross-sensitization to nicotine and other behavioral adaptations in adulthood in C57BL/6J and DBA/2J mice.

Adolescent alcohol exposure is associated with lasting behavioral changes in humans and in mice. Prior work from our laboratory and others have demonstrated that C57BL/6J and DBA/2J mice differ in sensitivity to some effects of acute alcohol exposure during adolescence and adulthood. However, it is unknown if these strains differ in cognitive, anxiety-related, and addiction-related long-term consequences of adolescent intermittent alcohol exposure. This study examined the impact of a previously validated adolescent alcohol exposure paradigm (2-3 g/kg, i.p., every other day PND 30-44) in C57BL/6J and DBA/2J male and female mice on adult fear conditioning, anxiety-related behavior (elevated plus maze), and addiction-related phenotypes including nicotine sensitivity (hypothermia and locomotor depression) and alcohol sensitivity (loss of righting reflex; LORR). Both shared and strain-specific long-term consequences of adolescent alcohol exposure were found. Most notably, we found a strain-specific alcohol-induced increase in sensitivity to nicotine's hypothermic effects during adulthood in the DBA/2J strain but not in the C57BL/6J strain. Conversely, both strains demonstrated a robust increased latency to LORR during adulthood after adolescent alcohol exposure. Thus, we observed strain-dependent cross-sensitization to nicotine and strain-independent tolerance to alcohol due to adolescent alcohol exposure. Several strain and sex differences independent of adolescent alcohol treatment were also observed. These include increased sensitivity to nicotine-induced hypothermia in the C57BL/6J strain relative to the DBA/2J strain, in addition to DBA/2J mice showing more anxiety-like behaviors in the elevated plus maze relative to the C57BL/6J strain. Overall, these results suggest that adolescent alcohol exposure results in altered adult sensitivity to nicotine and alcohol with some phenotypes mediated by genetic background.

Long-term safety and dose escalation of intracerebroventricular CLN5 gene therapy in sheep supports clinical translation for CLN5 Batten disease.

CLN5 neuronal ceroid lipofuscinosis (NCL, Batten disease) is a rare, inherited fatal neurodegenerative disorder caused by mutations in the CLN5 gene. The disease is characterised by progressive neuronal loss, blindness, and premature death. There is no cure. This study evaluated the efficacy of intracerebroventricular (ICV) delivery of an adeno-associated viral vector encoding ovine CLN5 (scAAV9/oCLN5) in a naturally occurring sheep model of CLN5 disease. CLN5 affected (CLN5-/-) sheep received low, moderate, or high doses of scAAV9/oCLN5 at three disease stages. The treatment delayed disease progression, extended survival and slowed stereotypical brain atrophy in most animals. Of note, one high dose treated animal only developed mild disease symptomology and survived to 60.1 months of age, triple the natural life expectancy of an untreated CLN5-/- sheep. Eyesight was not preserved at any administration age or dosage. Histopathologic examination revealed that greater transduction efficiency was achieved through higher ICV doses, and this resulted in greater amelioration of disease pathology. Together with other pre-clinical data from CLN5-/- sheep, the safety and efficacy data from these investigational new drug (IND)-enabling studies supported the initiation of the first in-human CLN5 gene therapy clinical study using the ICV delivery route for the treatment of CLN5 NCL. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT05228145.

Intraparenchymal convection enhanced delivery of AAV in sheep to treat Mucopolysaccharidosis IIIC.

BACKGROUND: Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme, heparan sulfate acetyl-CoA: α-glucosaminide-N-acetyltransferase (HGSNAT), is bound to the lysosomal membrane, therefore cannot cross the blood-brain barrier or diffuse between cells. We previously demonstrated disease correction in MPSIIIC mice using an Adeno-Associated Vector (AAV) delivering HGSNAT via intraparenchymal brain injections using an AAV2 derived AAV-truetype (AAV-TT) serotype with improved distribution over AAV9. METHODS: Here, intraparenchymal AAV was delivered in sheep using catheters or Hamilton syringes, placed using Brainlab cranial navigation for convection enhanced delivery, to reduce proximal vector expression and improve spread. RESULTS: Hamilton syringes gave improved AAV-GFP distribution, despite lower vector doses and titres. AAV-TT-GFP displayed moderately better transduction compared to AAV9-GFP but both serotypes almost exclusively transduced neurons. Functional HGSNAT enzyme was detected in 24-37% of a 140g gyrencephalic sheep brain using AAV9-HGSNAT with three injections in one hemisphere. CONCLUSIONS: Despite variabilities in volume and titre, catheter design may be critical for efficient brain delivery. These data help inform a clinical trial for MPSIIIC.

Infant externalizing behavior and parent depressive symptoms: Prospective predictors of parental pandemic related distress.

Understanding predictors and effects of the COVID-19 Pandemic is a top-priority in research endeavors. The impact of COVID-19 on all components of family life and mental health cannot be overstated. This study emphasizes the need to investigate predictors of parents' responses to disaster by conceptualizing the depth of the impact of the pandemic using Bronfenbrenner's Bioecological Systems Model. We evaluate parents of infants as the center of the microsystem and discuss the importance of parents' responses to the pandemic for children's development. Specifically, utilizing a prospective design involving a sample of 105 infant-mother-father triads, we test the predictive effects of mothers' and fathers' mental health and infant externalizing behavior assessed prior to the pandemic when infants were 16-months on later pandemic related distress (PRD) approximately 1 year later. Results indicate that for both mothers and fathers, more depressive symptoms during their child's infancy predicted more PRD. Although mothers' reports of more child externalizing behavior significantly predicted more PRD, fathers' reports of externalizing were strongly, positively correlated with their concurrent depressive symptoms but not directly related to PRD. We demonstrate the importance of pre-existing mental health and parents' perceptions of their children's behavior as early as 16 months, in coping with disaster.

Progressive MRI brain volume changes in ovine models of CLN5 and CLN6 neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (Batten disease) are a group of inherited lysosomal storage disorders characterized by progressive neurodegeneration leading to motor and cognitive dysfunction, seizure activity and blindness. The disease can be caused by mutations in 1 of 13 ceroid lipofuscinosis neuronal (CLN) genes. Naturally occurring sheep models of the CLN5 and CLN6 neuronal ceroid lipofuscinoses recapitulate the clinical disease progression and post-mortem pathology of the human disease. We used longitudinal MRI to assess global and regional brain volume changes in CLN5 and CLN6 affected sheep compared to age-matched controls over 18 months. In both models, grey matter volume progressively decreased over time, while cerebrospinal fluid volume increased in affected sheep compared with controls. Total grey matter volume showed a strong positive correlation with clinical scores, while cerebrospinal fluid volume was negatively correlated with clinical scores. Cortical regions in affected animals showed significant atrophy at baseline (5 months of age) and progressively declined over the disease course. Subcortical regions were relatively spared with the exception of the caudate nucleus in CLN5 affected animals that degenerated rapidly at end-stage disease. Our results, which indicate selective vulnerability and provide a timeline of degeneration of specific brain regions in two sheep models of neuronal ceroid lipofuscinoses, will provide a clinically relevant benchmark for assessing therapeutic efficacy in subsequent trials of gene therapy for CLN5 and CLN6 disease.

Associations Between Children's Emotion Regulation, Mindful Parenting, Parent Stress, and Parent Coping During the COVID-19 Pandemic.

Stress among parents has increased during the COVID-19 pandemic. Research prior to the pandemic indicates that parents of children who struggle with emotion regulation (ER) and who themselves are less mindful report more stress and diminished coping abilities. We know little, however, about these associations in the context of COVID-19. To prevent COVID-related deteriorations in parent well-being and child outcomes and to support parents during this potentially challenging time, it is important to understand the factors that are associated with increased stress as well as adaptive coping. This paper discusses the association between children's ER, mindful parenting (MP), parent stress, and parents' coping with parenting during the pandemic in a sample of 217 caregivers of school-aged children (91.0% mothers). Results indicated that children's ER was associated with parents' self-reported coping with parenting in the pandemic but was not associated with increased stress. Further, MP moderated the association between children's ER and coping, such that parents who were the most mindful and had children with better ER skills reported significantly greater ability to cope with pandemic parenting. Coping was lower for other combinations of ER and mindful parenting. These findings contradict those from before COVID, suggesting the relationship between children's ER and parent outcomes may differ in the COVID-19 context, and offering insights into which parents may be most likely to struggle with coping with pandemic parenting.

Intravitreal Injections in the Ovine Eye.

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that fills the posterior chamber of the eye and maintains the shape of the eye globe. Although the IVT route is less specifically targeted than subretinal delivery, it is much less invasive and is widely used in clinical settings for a range of ocular diseases. We previously demonstrated the efficacy of intravitreal delivery of an adeno-associated virus (AAV)-mediated gene therapy product (AAV9.CLN5) in sheep with a naturally occurring CLN5 form of neuronal ceroid lipofuscinosis (NCL). Affected sheep received IVT gene therapy in one eye, with the other untreated eye serving as an internal control. Retinal structure and function were maintained in the treated eye up to 15 months after treatment, while the untreated eye displayed progressively declining function and severe atrophy during postmortem examination. Based on the sheep studies, the CLN5 gene therapy product was cleared as a candidate investigational new drug (IND) by the United States Food and Drug Administration in September 2021. This paper details the surgical protocol for IVT delivery of a therapeutic viral vector to the ovine eye.

Aggregation chimeras provide evidence of in vivo intercellular correction in ovine CLN6 neuronal ceroid lipofuscinosis (Batten disease).

The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are fatal, mainly childhood, inherited neurodegenerative lysosomal storage diseases. Sheep affected with a CLN6 form display progressive regionally defined glial activation and subsequent neurodegeneration, indicating that neuroinflammation may be causative of pathogenesis. In this study, aggregation chimeras were generated from homozygous unaffected normal and CLN6 affected sheep embryos, resulting in seven chimeric animals with varied proportions of normal to affected cells. These sheep were classified as affected-like, recovering-like or normal-like, based on their cell-genotype ratios and their clinical and neuropathological profiles. Neuropathological examination of the affected-like animals revealed intense glial activation, prominent storage body accumulation and severe neurodegeneration within all cortical brain regions, along with vision loss and decreasing intracranial volumes and cortical thicknesses consistent with ovine CLN6 disease. In contrast, intercellular communication affecting pathology was evident at both the gross and histological level in the normal-like and recovering-like chimeras, resulting in a lack of glial activation and rare storage body accumulation in only a few cells. Initial intracranial volumes of the recovering-like chimeras were below normal but progressively recovered to about normal by two years of age. All had normal cortical thicknesses, and none went blind. Extended neurogenesis was evident in the brains of all the chimeras. This study indicates that although CLN6 is a membrane bound protein, the consequent defect is not cell intrinsic. The lack of glial activation and inflammatory responses in the normal-like and recovering-like chimeras indicate that newly generated cells are borne into a microenvironment conducive to maturation and survival.

The Translational Benefits of Sheep as Large Animal Models of Human Neurological Disorders.

The past two decades have seen a considerable rise in the use of sheep to model human neurological disorders. While each animal model has its merits, sheep have many advantages over small animal models when it comes to studies on the brain. In particular, sheep have brains more comparable in size and structure to the human brain. They also have much longer life spans and are docile animals, making them useful for a wide range of in vivo studies. Sheep are amenable to regular blood and cerebrospinal fluid sampling which aids in biomarker discovery and monitoring of treatment efficacy. Several neurological diseases have been found to occur naturally in sheep, however sheep can also be genetically engineered or experimentally manipulated to recapitulate disease or injury. Many of these types of sheep models are currently being used for pre-clinical therapeutic trials, particularly gene therapy, with studies from several models culminating in potential treatments moving into clinical trials. This review will provide an overview of the benefits of using sheep to model neurological conditions, and highlight naturally occurring and experimentally induced sheep models that have demonstrated translational validity.

Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease.

Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of inherited neurodegenerative diseases primarily affecting children. A common feature across most NCLs is the progressive loss of vision. We performed intravitreal injections of self-complementary AAV9 vectors packaged with either ovine CLN5 or CLN6 into one eye of 3-month-old CLN5-/- or CLN6-/- animals, respectively. Electroretinography (ERG) was performed every month following treatment, and retinal histology was assessed post-mortem in the treated compared to untreated eye. In CLN5-/- animals, ERG amplitudes were normalised in the treated eye whilst the untreated eye declined in a similar manner to CLN5 affected controls. In CLN6-/- animals, ERG amplitudes in both eyes declined over time although the treated eye showed a slower decline. Post-mortem examination revealed significant attenuation of retinal atrophy and lysosomal storage body accumulation in the treated eye compared with the untreated eye in CLN5-/- animals. This proof-of-concept study provides the first observation of efficacious intravitreal gene therapy in a large animal model of NCL. In particular, the single administration of AAV9-mediated intravitreal gene therapy can successfully ameliorate retinal deficits in CLN5-/- sheep. Combining ocular gene therapy with brain-directed therapy presents a promising treatment strategy to be used in future sheep trials aiming to halt neurological and retinal disease in CLN5 Batten disease.

Inhibition of Invasion by Polyphenols from Citrus Fruit and Berries in Human Malignant Glioma Cells In Vitro.

BACKGROUND/AIM: The outlook for patients with high grade glioma (HGG) remains dismal. Hence, attention has focused on numerous innovative treatments. Our group has proposed a strategy on the use of a combination of polyphenols, as anti-invasive agents for the management of these neoplasms. MATERIALS AND METHODS: The aim of this study was to evaluate the in vitro effects of citrus flavonoids (tangeretin, nobiletin, naringin and limonin) and berry extracts (chokeberry, elderberry and bilberry) on selected mediators of invasion in 2 HGG cell cultures. RESULTS: The IC50 values could only be determined for tangeretin and chokeberry extract. The rest were non-functional in this context. Immunocytochemistry and flow cytometry results showed that chokeberry extract was most effective in down-regulating the expression of CD44. Similarly, RT-PCR data supported its ability to reduce gene expression of MMP-14 and EGFR. 2D invasion assays confirmed that inhibition is greater with chokeberry extract. CONCLUSION: Both polyphenols have anti-invasive potential but chokeberry extract is a stronger agent for glioma management.

Long-term participation in collaborative fisheries research improves angler opinions on marine protected areas.

Recent marine spatial planning efforts, including the management and monitoring of marine protected areas (MPAs), increasingly focus on the importance of stakeholder engagement. For nearly 15 years, the California Collaborative Fisheries Research Program (CCFRP) has partnered volunteer anglers with researchers, the fishing industry, and resource managers to monitor groundfishes in California's network of MPAs. While the program has succeeded in generating sustained biological observations, we know little about volunteer angler demography or the impact of participation on their perceptions and opinions on fisheries data or MPAs. In this study we surveyed CCFRP volunteers to learn about (a) volunteer angler demographics and attitudes toward groundfish management and stock health, (b) volunteer angler motivations for joining and staying in the program, and (c) whether participation in the program influenced volunteer angler opinions on the quality of fisheries data used in resource management and the establishment of MPAs in California. CCFRP volunteers were older and had higher fishing avidity than average within the California recreational angling community. Many self-identified as more conservation-minded than their peers in the recreational fishing community and had positive views of California groundfish management and stock health. Participation in science and giving back to fisheries resources were major motivating factors in their decision to become and remain CCFRP volunteers. Angler opinions toward MPAs were more positive after volunteering with CCFRP. Those who had volunteered for seven or more years with CCFRP were more likely than not to gain a positive opinion of MPAs. Our survey results provide evidence that long-term engagement of stakeholders in collaborative research positively influences stakeholder opinions regarding marine resource management, and highlights CCFRP's success in engaging citizen science stakeholders in collaborative fisheries research.

Chemical neuroanatomy of the substantia nigra in the ovine brain.

The substantia nigra is an integral component of the basal ganglia circuitry for limbic and motor functions. Dysfunction and degeneration of the basal ganglia are fundamental aspects of neurodegenerative diseases such as Parkinson's disease and Huntington's disease. With the increasing use of sheep to model neurological diseases, it is crucial to understand the anatomy and neurochemistry of these key basal ganglia nuclei in the normal sheep brain and how they compare to the human brain. Therefore, studies of the gross anatomy, cellular morphology, and neurochemical expression patterns within the sheep substantia nigra were performed. We show that the sheep substantia nigra reflects all important aspects of the anatomy and neurochemistry of the human substantia nigra, with only minor inter-species differences evident. Many neurochemicals that are central to the functioning of the SN, and wider basal ganglia circuitry, are present throughout the sheep SN. In a wider context, the results of this study provide evidence that the sheep substantia nigra accurately reflects the anatomy of the human substantia nigra, which validates the use of sheep models of basal ganglia neurological disorders.

Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens.

The Gram-positive pathogen Clostridium perfringens possesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present in C. perfringens, called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of the pilB2 and pilC2 genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detected in vivo or in vitro even in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase from C. perfringens (CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levels in vivo but that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously from in vivo studies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium.IMPORTANCE Type IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable species Clostridium perfringens was used to study proteins involved in polymerizing the pilin, PilA2, into a pilus. The assembly ATPase PilB2 and its cognate membrane protein partner, PilC2, were purified. PilB2 bound the intracellular signal molecule c-di-GMP. Increased levels of intracellular c-di-GMP led to increased polymerization of PilA2, indicating that Gram-positive bacteria use this molecule to regulate pilus synthesis. These findings provide valuable information for understanding how pathogenic clostridia regulate TFP to cause human diseases.

Associations between bride price obligations and women's anger, symptoms of mental distress, poverty, spouse and family conflict and preoccupations with injustice in conflict-affected Timor-Leste.

OBJECTIVES: Bride price is a widespread custom in many parts of the world, including in most countries in sub-Saharan Africa and parts of Asia. We hypothesised that problems relating to the obligatory ongoing remittances made by the husband and his family to the bride's family may be a source of mental disturbance (in the form of explosive anger and severe mental distress) among women. In addition, we postulated that problems arising with bride price would be associated with conflict with the spouse and family, poverty and women's preoccupations with injustice. DESIGN: A mixed-methods study comprising a total community household survey and semistructured qualitative interviews. SETTING: Two villages, one urban, the other rural, in Timor-Leste. PARTICIPANTS: 1193 married women participated in the household survey and a structured subsample of 77 women participated in qualitative interviews. RESULTS: Problems with bride price showed a consistent dose-effect relationship with sudden episodes of explosive anger, excessive anger and severe psychological distress. Women with the most severe problems with bride price had twice the poverty scores as those with no problems with the custom. Women with the most severe problems with bride price also reported a threefold increase in conflict with their spouse and a fivefold increase in conflict with family. They also reported heightened preoccupations with injustice. CONCLUSIONS: Our study is the first to show consistent associations between problems with bride price obligations and mental distress, poverty, conflict with spouse and family and preoccupations with injustice among women in a low-income, postconflict country.

Auranofin is an apoptosis-simulating agent with in vitro and in vivo anti-leishmanial activity.

Cutaneous leishmaniasis remains ignored in therapeutic drug discovery programs worldwide. This is mainly because cutaneous leishmaniasis is frequently a disease of impoverished populations in countries where funds are limited for research and patient care. However, the health burden of individuals in endemic areas mandates readily available, effective, and safe treatments. Of the existing cutaneous leishmaniasis therapeutics, many are growth inhibitory to Leishmania parasites, potentially creating dormant parasite reservoirs that can be activated when host immunity is compromised, enabling the reemergence of cutaneous leishmaniasis lesions or worse spread of Leishmania parasites to other body sites. To accelerate the identification and development of novel cutaneous leishmaniasis therapeutics, we designed an integrated in vitro and in vivo screening platform that incorporated multiple Leishmania life cycles and species and probed a focused library of pharmaceutically active compounds. The objective of this phenotypic drug discovery platform was the identification and prioritization of bona fide cytotoxic chemotypes toward Leishmania parasites. We identified the Food and Drug Administration-approved drug auranofin, a known inhibitor of Leishmania promastigote growth, as a potent cytotoxic anti-leishmanial agent and inducer of apoptotic-like death in promastigotes. Significantly, the anti-leishmanial activity of auranofin transferred to cell-based amastigote assays as well as in vivo murine models. With appropriate future investigation, these data may provide the foundation for potential exploitation of gold(I)-based complexes as chemical tools or the basis of therapeutics for leishmaniasis. Thus, auranofin may represent a prototype drug that can be used to identify signaling pathways within the parasite and host cell critical for parasite growth and survival.

Effect of thyroid hormone T3 on myosin-Va expression in the central nervous system.

Thyroid hormones (THs) are essential for brain development, where they regulate gliogenesis, myelination, cell proliferation and protein synthesis. Hypothyroidism severely affects neuronal growth and establishment of synaptic connections. Triiodothyronine (T3), the biologically active form of TH, has a central function in these activities. So, Myosin-Va (Myo-Va), a molecular motor protein involved in vesicle and RNA transport, is a good candidate as a target for T3 regulation. Here, we analyzed Myo-Va expression in euthyroid and hypothyroid adult rat brains and synaptosomes. We observed a reduction of Myo-Va expression in cultured neural cells from newborn hypothyroid rat brain, while immunocytochemical experiments showed a punctate distribution of this protein in the cytoplasm of cells. Particularly, Myo-Va co-localized with microtubules in neurites, especially in their varicosities. Myo-Va immunostaining was stronger in astrocytes and neurons of controls when compared with hypothyroid brains. In addition, supplementation of astrocyte cultures with T3 led to increased expression of Myo-Va in cells from both euthyroid and hypothyroid animals, suggesting that T3 modulates Myo-Va expression in neural cells both in vivo and in vitro. We have further analyzed Myo-Va expression in U373 cells, a human glioblastoma line, and found the same punctate cytoplasmic protein localization. As in normal neural cells, this expression was also increased by T3, suggesting that the modulatory mechanism exerted by T3 over Myo-Va remains active on astrocyte tumor cells. These data, coupled with the observation that Myo-Va is severely affected in hypothyroidism, support the hypothesis that T3 activity regulates neural motor protein expression, taking Myo-Va as a model. As a consequence, reduced T3 activity could supposedly affect axonal transport and synaptic function, and could therefore explain disturbances seen in the hypothyroid brain.

Approaches to mitochondrially mediated cancer therapy.

For some malignant cancers even combined surgical, radiotherapeutic and chemotherapeutic approaches are not curative, indeed, in certain tumour types even a modest survival benefit is difficult to achieve. There are various biological reasons which underlie this profound resistance but the propensity of cancer cells to repair breaks caused by DNA-damaging radiation and cytotoxic drugs is of major significance in this context. Such highly resistant tumours include the malignant gliomas which are intrinsic to and directly affect the brain and spinal cord. In evaluating approaches which do not elicit tumour cell death directly by DNA damage, it is intriguing to consider mitochondrially mediated apoptosis as a potentially effective alternative. Since the mitochondrial membrane potentials in cancer cells are frequently reduced in comparison with those of non-neoplastic cells this allows a window of opportunity for small molecule agents to enter the tumour cell mitochondria and reduce oxygen consumption with subsequent release of cytochrome c and activation of a caspase pathway to apoptosis which is cancer cell specific. In the quest for agents which can selectively destroy neoplastic cells in this manner, whilst leaving normal adjacent cells intact, various tricyclic drugs have come under scrutiny. In a range of laboratory assays we, and others, have established that certain cancers and, in particular, malignant glioma, are intrinsically sensitive to this approach. We have also established the cellular, molecular and biochemical mechanisms underlying this process. While such archival tricyclics as the antidepressants, clomipramine and amitriptyline, have been used in these experiments their commercial development in cancer therapy has not been forthcoming and their clinical use in glioma has been confined to anecdotal cases. In addition, the dose-dependant role of agents such as anticonvulsants and steroids commonly used in glioma patients in modulating efficacy of the tricyclics is a matter for continued investigation. Other ways of targeting the mitochondrion for cancer therapy include exploitation of the 18kDa translocator protein (peripheral-type benzodiazepine receptor) within the mitochondrial permeability transition pore and enzymatic or molecular modification of a species of ganglioside (GD3/GD3(A)) expressed on the surface of neoplastic cells which are determinants of mitochondrially mediated apoptosis. It is hoped that such approaches may lead to clinical programmes which will improve the prognosis for patients suffering from highly resistant neoplasms.