Stanislav S. Zakharenko

Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation

Cancer stem cells are remarkably similar to normal stem cells: both self-renew, are multipotent and express common surface markers, for example, prominin 1 (PROM1, also called CD133). What remains unclear is whether cancer stem cells are the direct progeny of mutated stem cells or more mature cells that reacquire stem cell properties during tumour formation. Answering this question will require knowledge of whether normal stem cells are susceptible to cancer-causing mutations; however, this has proved difficult to test because the identity of most adult tissue stem cells is not known. Here, using an inducible Cre, nuclear LacZ reporter allele knocked into the Prom1 locus (Prom1C-L), we show that Prom1 is expressed in a variety of developing and adult tissues. Lineage-tracing studies of adult Prom1+/C-L mice containing the Rosa26-YFP reporter allele showed that Prom1+ cells are located at the base of crypts in the small intestine, co-express Lgr5 (ref. 2), generate the entire intestinal epithelium, and are therefore the small intestinal stem cell. Prom1 was reported recently to mark cancer stem cells of human intestinal tumours that arise frequently as a consequence of aberrant wingless (Wnt) signalling. Activation of endogenous Wnt signalling in Prom1+/C-L mice containing a Cre-dependent mutant allele of β-catenin (Ctnnb1lox(ex3)) resulted in a gross disruption of crypt architecture and a disproportionate expansion of Prom1+ cells at the crypt base. Lineage tracing demonstrated that the progeny of these cells replaced the mucosa of the entire small intestine with neoplastic tissue that was characterized by focal high-grade intraepithelial neoplasia and crypt adenoma formation. Although all neoplastic cells arose from Prom1+ cells in these mice, only 7% of tumour cells retained Prom1 expression. Our data indicate that Prom1 marks stem cells in the adult small intestine that are susceptible to transformation into tumours retaining a fraction of mutant Prom1+ tumour cells.

Transcriptional and behavioral interaction between 22q11.2 orthologs modulates schizophrenia-related phenotypes in mice

Microdeletions of 22q11.2 represent one of the highest known genetic risk factors for schizophrenia. It is likely that more than one gene contributes to the marked risk associated with this locus. Two of the candidate risk genes encode the enzymes proline dehydrogenase (PRODH) and catechol-O-methyltransferase (COMT), which modulate the levels of a putative neuromodulator (L-proline) and the neurotransmitter dopamine, respectively. Mice that model the state of PRODH deficiency observed in humans with schizophrenia show increased neurotransmitter release at glutamatergic synapses as well as deficits in associative learning and response to psychomimetic drugs. Transcriptional profiling and pharmacological manipulations identified a transcriptional and behavioral interaction between the Prodh and Comt genes that is likely to represent a homeostatic response to enhanced dopaminergic signaling in the frontal cortex. This interaction modulates a number of schizophrenia-related phenotypes, providing a framework for understanding the high disease risk associated with this locus, the expression of the phenotype, or both.

FMRP Regulates Neurotransmitter Release and Synaptic Information Transmission by Modulating Action Potential Duration via BK Channels

Loss of FMRP causes fragile X syndrome (FXS), but the physiological functions of FMRP remain highly debatable. Here we show that FMRP regulates neurotransmitter release in CA3 pyramidal neurons by modulating action potential (AP) duration. Loss of FMRP leads to excessive AP broadening during repetitive activity, enhanced presynaptic calcium influx, and elevated neurotransmitter release. The AP broadening defects caused by FMRP loss have a cell-autonomous presynaptic origin and can be acutely rescued in postnatal neurons. These presynaptic actions of FMRP are translation independent and are mediated selectively by BK channels via interaction of FMRP with BK channels regulatory β4 subunits. Information-theoretical analysis demonstrates that loss of these FMRP functions causes marked dysregulation of synaptic information transmission. FMRP-dependent AP broadening is not limited to the hippocampus, but also occurs in cortical pyramidal neurons. Our results thus suggest major translation-independent presynaptic functions of FMRP that may have important implications for understanding FXS neuropathology.

Differentiated Horizontal Interneurons Clonally Expand to Form Metastatic Retinoblastoma in Mice

During neurogenesis, the progression from a progenitor cell to a differentiated neuron is believed to be unidirectional and irreversible. The Rb family of proteins (Rb, p107, and p130) regulates cell-cycle exit and differentiation during retinogenesis. Rb and p130 are redundantly expressed in the neurons of the inner nuclear layer (INL) of the retina. We have found that in the adult Rb;p130-deficient retinae p107 compensation prevents ectopic proliferation of INL neurons. However, p107 is haploinsufficient in this process. Differentiated Rb(-/-);p107(+/-);p130(-/-) horizontal interneurons re-entered the cell cycle, clonally expanded, and formed metastatic retinoblastoma. Horizontal cells were not affected in Rb(+/-);p107(-/-);p130(-/-) or Rb(-/-);p107(-/-);p130(+/-), retinae suggesting that one copy of Rb or p130 was sufficient to prevent horizontal proliferation. We hereby report that differentiated neurons can proliferate and form cancer while maintaining their differentiated state including neurites and synaptic connections.

Phosphatase and tensin homolog, deleted on chromosome 10 deficiency in brain causes defects in synaptic structure, transmission and plasticity, and myelination abnormalities

The phosphatidylinositol 3-kinase (PI3K) signaling pathway modulates growth, proliferation and cell survival in diverse tissue types and plays specialized roles in the nervous system including influences on neuronal polarity, dendritic branching and synaptic plasticity. The tumor-suppressor phosphatase with tensin homology (PTEN) is the central negative regulator of the PI3K pathway. Germline PTEN mutations result in cancer predisposition, macrocephaly and benign hamartomas in many tissues, including Lhermitte-Duclos disease, a cerebellar growth disorder. Neurological abnormalities including autism, seizures and ataxia have been observed in association with inherited PTEN mutation with variable penetrance. It remains unclear how loss of PTEN activity contributes to neurological dysfunction. To explore the effects of Pten deficiency on neuronal structure and function, we analyzed several ultra-structural features of Pten-deficient neurons in Pten conditional knockout mice. Using Golgi stain to visualize full neuronal morphology, we observed that increased size of nuclei and somata in Pten-deficient neurons was accompanied by enlarged caliber of neuronal projections and increased dendritic spine density. Electron microscopic evaluation revealed enlarged abnormal synaptic structures in the cerebral cortex and cerebellum. Severe myelination defects included thickening and unraveling of the myelin sheath surrounding hypertrophic axons in the corpus callosum. Defects in myelination of axons of normal caliber were observed in the cerebellum, suggesting intrinsic abnormalities in Pten-deficient oligodendrocytes. We did not observe these abnormalities in wild-type or conditional Pten heterozygous mice. Moreover, conditional deletion of Pten drastically weakened synaptic transmission and synaptic plasticity at excitatory synapses between CA3 and CA1 pyramidal neurons in the hippocampus. These data suggest that Pten is involved in mechanisms that control development of neuronal and synaptic structures and subsequently synaptic function.

Slow Presynaptic and Fast Postsynaptic Components of Compound Long-Term Potentiation

Long-term potentiation (LTP) mediates learning and memory in the mammalian hippocampus. Whether a presynaptic or postsynaptic neuron principally enhances synaptic transmission during LTP remains controversial. Acute hippocampal slices were made from transgenic mouse strains that express synaptopHluorin in neurons. SynaptopHluorin is an indicator of synaptic vesicle recycling; thus, we monitored functional changes in presynaptic boutons of CA3 pyramidal cells by measuring changes in synaptopHluorin fluorescence. Simultaneously, we recorded field excitatory postsynaptic potentials to monitor changes in the strength of excitatory synapses between CA3 and CA1 pyramidal neurons. We found that LTP consists of two components, a slow presynaptic component and a fast postsynaptic component. The presynaptic mechanisms contribute mostly to the late phase of compound LTP, whereas the postsynaptic mechanisms are crucial during the early phase of LTP. We also found that protein kinase A (PKA) and L-type voltage-gated calcium channels are crucial for the expression of the presynaptic component of compound LTP, and NMDA channels are essential for that of the postsynaptic component of LTP. These data are the first direct evidence that presynaptic and postsynaptic components of LTP are temporally and mechanistically distinct.

Connectivity Patterns Revealed by Mapping of Active Inputs on Dendrites of Thalamorecipient Neurons in the Auditory Cortex

Despite being substantially outnumbered by intracortical inputs on thalamorecipient neurons, thalamocortical projections efficiently deliver acoustic information to the auditory cortex. We hypothesized that thalamic projections may achieve effectiveness by forming synapses at optimal locations on dendritic trees of cortical neurons. Using two-photon calcium imaging in dendritic spines, we constructed maps of active thalamic and intracortical inputs on dendritic trees of thalamorecipient cortical neurons in mouse thalamocortical slices. These maps revealed that thalamic projections synapse preferentially on stubby dendritic spines within 100 μm of the soma, whereas the locations and morphology of spines that receive intracortical projections have a less-defined pattern. Using two-photon photolysis of caged glutamate, we found that activation of stubby dendritic spines located perisomatically generated larger postsynaptic potentials in the soma of thalamorecipient neurons than did activation of remote dendritic spines or spines of other morphological types. These results suggest a novel mechanism of reliability of thalamic projections: the positioning of crucial afferent inputs at optimal synaptic locations.

Dissecting the Components of Long-Term Potentiation

The formation of memories relies in part on plastic changes at synapses between neurons. Although the mechanisms of synaptic plasticity have been studied extensively over several decades, many aspects of this process remain controversial. The cellular locus of expression of long-term potentiation (LTP), a major form of synaptic plasticity, is one of the most important unresolved phenomena. In this article, some recent advances in this area made possible by the development of new imaging tools are summarized. These studies have demonstrated that LTP is compound in nature and consists of both presynaptic and postsynaptic components. Some features of presynaptic and postsynaptic changes during compound LTP are also reviewed.

Age-dependent microRNA control of synaptic plasticity in 22q11 deletion syndrome and schizophrenia.

The 22q11 deletion syndrome (22q11DS) is characterized by multiple physical and psychiatric abnormalities and is caused by the hemizygous deletion of a 1.5–3 Mb region of chromosome 22. It constitutes one of the strongest known genetic risks for schizophrenia; schizophrenia arises in as many as 30% of patients with 22q11DS during adolescence or early adulthood. A mouse model of 22q11DS displays an age-dependent increase in hippocampal long-term potentiation (LTP), a form of synaptic plasticity underlying learning and memory. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2), which is responsible for loading Ca2+ into the endoplasmic reticulum (ER), is elevated in this mouse model. The resulting increase in ER Ca2+ load leads to enhanced neurotransmitter release and increased LTP. However, the mechanism by which the 22q11 microdeletion leads to SERCA2 overexpression and LTP increase has not been determined. Screening of multiple mutant mouse lines revealed that haploinsufficiency of Dgcr8, a microRNA (miRNA) biogenesis gene in the 22q11DS disease-critical region, causes age-dependent, synaptic SERCA2 overexpression and increased LTP. We found that miR-25 and miR-185, regulators of SERCA2, are depleted in mouse models of 22q11DS. Restoration of these miRNAs to presynaptic neurons rescues LTP in Dgcr8+/− mice. Finally, we show that SERCA2 is elevated in the brains of patients with schizophrenia, providing a link between mouse model findings and the human disease. We conclude that miRNA-dependent SERCA2 dysregulation is a pathogenic event in 22q11DS and schizophrenia.

Phosphatase and tensin homologue (PTEN) regulates synaptic plasticity independently of its effect on neuronal morphology and migration

Non‐technical summary  The sizes of neurons and their synaptic connections are regulated by multiple molecular mechanisms to provide neuronal networks that perform well‐defined functions. Deletion of the tumour suppressor phosphatase and tensin homologue (PTEN) during early development leads to a 2‐ to 3‐fold increase in neuronal and synaptic size and abnormalities in synaptic plasticity, the cellular mechanism underlying learning and memory. Whether PTEN deletion affects synaptic plasticity directly or as a consequence of its effect on the neuronal and synaptic size remained unclear. Here we show that deletion of the Pten gene Pten in mice during postnatal development, when the central nervous system is formed, does not affect neuronal or synaptic size but impairs synaptic plasticity. Thus, PTEN affects neuronal structure and synaptic plasticity through independent mechanisms.