Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice.

Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

Neuronal Tsc1/2 complex controls autophagy through AMPK-dependent regulation of ULK1.

Tuberous sclerosis complex (TSC) is a disorder arising from mutation in the TSC1 or TSC2 gene, characterized by the development of hamartomas in various organs and neurological manifestations including epilepsy, intellectual disability and autism. TSC1/2 protein complex negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) a master regulator of protein synthesis, cell growth and autophagy. Autophagy is a cellular quality-control process that sequesters cytosolic material in double membrane vesicles called autophagosomes and degrades it in autolysosomes. Previous studies in dividing cells have shown that mTORC1 blocks autophagy through inhibition of Unc-51-like-kinase1/2 (ULK1/2). Despite the fact that autophagy plays critical roles in neuronal homeostasis, little is known on the regulation of autophagy in neurons. Here we show that unlike in non-neuronal cells, Tsc2-deficient neurons have increased autolysosome accumulation and autophagic flux despite mTORC1-dependent inhibition of ULK1. Our data demonstrate that loss of Tsc2 results in autophagic activity via AMPK-dependent activation of ULK1. Thus, in Tsc2-knockdown neurons AMPK activation is the dominant regulator of autophagy. Notably, increased AMPK activity and autophagy activation are also found in the brains of Tsc1-conditional mouse models and in cortical tubers resected from TSC patients. Together, our findings indicate that neuronal Tsc1/2 complex activity is required for the coordinated regulation of autophagy by AMPK. By uncovering the autophagy dysfunction associated with Tsc2 loss in neurons, our work sheds light on a previously uncharacterized cellular mechanism that contributes to altered neuronal homeostasis in TSC disease.

A randomized, double-blind, placebo controlled, parallel group, efficacy study of alpha BRAIN® administered orally.

OBJECTIVE: Alpha BRAIN® is a nootropic supplement that purports to enhance cognitive functioning in healthy adults. The goal of this study was to investigate the efficacy of this self-described cognitive enhancing nootropic on cognitive functioning in a group of healthy adults by utilizing a randomized, double blind, placebo-controlled design. METHODS: A total of 63-treatment naïve individuals between 18 and 35 years of age completed the randomized, double-blind, placebo controlled trial. All participants completed a 2-week placebo run in before receiving active product, Alpha BRAIN® or new placebo, for 6 weeks. Participants undertook a battery of neuropsychological tests at randomization and at study completion. Primary outcome measures included a battery of neuropsychological tests and measures of sleep. RESULTS: Compared with placebo, Alpha BRAIN® significantly improved on tasks of delayed verbal recall and executive functioning. Results also indicated significant time-by-group interaction in delayed verbal recall for the Alpha BRAIN® group. CONCLUSIONS: The use of Alpha BRAIN® for 6 weeks significantly improved recent verbal memory when compared with controls, in a group of healthy adults. While the outcome of the study is encouraging, this is the first randomized controlled trial of Alpha BRAIN®, and the results merit further study.

A vascular model of Tsc1 deficiency accelerates renal tumor formation with accompanying hemangiosarcomas.

UNLABELLED: Tuberous sclerosis complex (TSC) is an autosomal disease caused by inactivating mutations in either of the tumor suppressor genes TSC1 or TSC2. TSC-associated tumor growth is present in multiple tissues and organs including brain, kidney, liver, heart, lungs, and skin. In the kidney, TSC angiomyolipomas have aberrant vascular structures with abnormal endothelial cells, suggesting a role for endothelial mTORC1 function. In the current report, a genetically engineered mouse model (GEMM) with a conditional knockout allele of Tsc1 with a Darpp32-Cre allele displayed accelerated formation of both kidney cystadenomas and paw hemangiosarcomas. All mutant mice developed hemangiosarcomas on multiple paws by 6 weeks of age. By 16 weeks of age, the average mutant hind paw was 4.0 mm in diameter, nearly double the size of control mice. Furthermore, the hemangiosarcomas and kidney cystadenomas were responsive to intraperitoneal rapamycin treatment. Immunoblotting and immunostaining for phospho-S6 (pS6) and phospho-CAD showed that the effect of rapamycin on tumor size was through inhibition of the mTOR signaling pathway. Finally, elevated VEGF mRNA levels were also observed in hemangiosarcoma specimens. Because paw hemangiosarcomas are easily detectable and scorable for size and growth, this novel mouse model enables accelerated in vivo drug testing for therapies of TSC-related tumors. IMPLICATIONS: These findings provide a strong rationale for simultaneous use of this conditional knockout mouse as an in vivo genetic model while seeking new cancer therapies for TSC-related tumors.

Divergent dysregulation of gene expression in murine models of fragile X syndrome and tuberous sclerosis.

BACKGROUND: Fragile X syndrome and tuberous sclerosis are genetic syndromes that both have a high rate of comorbidity with autism spectrum disorder (ASD). Several lines of evidence suggest that these two monogenic disorders may converge at a molecular level through the dysfunction of activity-dependent synaptic plasticity. METHODS: To explore the characteristics of transcriptomic changes in these monogenic disorders, we profiled genome-wide gene expression levels in cerebellum and blood from murine models of fragile X syndrome and tuberous sclerosis. RESULTS: Differentially expressed genes and enriched pathways were distinct for the two murine models examined, with the exception of immune response-related pathways. In the cerebellum of the Fmr1 knockout (Fmr1-KO) model, the neuroactive ligand receptor interaction pathway and gene sets associated with synaptic plasticity such as long-term potentiation, gap junction, and axon guidance were the most significantly perturbed pathways. The phosphatidylinositol signaling pathway was significantly dysregulated in both cerebellum and blood of Fmr1-KO mice. In Tsc2 heterozygous (+/-) mice, immune system-related pathways, genes encoding ribosomal proteins, and glycolipid metabolism pathways were significantly changed in both tissues. CONCLUSIONS: Our data suggest that distinct molecular pathways may be involved in ASD with known but different genetic causes and that blood gene expression profiles of Fmr1-KO and Tsc2+/- mice mirror some, but not all, of the perturbed molecular pathways in the brain.