Defects in the cerebella of conditional Neurod1 null mice correlate with effective Tg(Atoh1-cre) recombination and granule cell requirements for Neurod1 for differentiation.

Neurod1 is a crucial basic helix-loop-helix gene for most cerebellar granule cells and mediates the differentiation of these cells downstream of Atoh1-mediated proliferation of the precursors. In Neurod1 null mice, granule cells die throughout the posterior two thirds of the cerebellar cortex during development. However, Neurod1 is also necessary for pancreatic beta-cell development, and therefore Neurod1 null mice are diabetic, which potentially influences cerebellar defects. Here, we report a new Neurod1 conditional knock-out mouse model created by using a Tg(Atoh1-cre) line to eliminate Neurod1 in the cerebellar granule cell precursors. Our data confirm and extend previous work on systemic Neurod1 null mice and show that, in the central lobules, granule cells can be eradicated in the absence of Neurod1. Granule cells in the anterior lobules are partially viable and depend on as yet unknown genes, but the Purkinje cells show defects not previously recognized. Interestingly, delayed and incomplete Tg(Atoh1-cre) upregulation occurs in the most posterior lobules; this leads to near normal expression of Neurod1 with a concomitant normal differentiation of granule cells, Purkinje cells, and unipolar brush cells in lobules IX and X. Our analysis suggests that Neurod1 negatively regulates Atoh1 to ensure a rapid transition from proliferative precursors to differentiating neurons. Our data have implications for research on medulloblastoma, one of the most frequent brain tumors of children, as the results suggest that targeted overexpression of Neurod1 under Atoh1 promoter control may initiate the differentiation of these tumors.

Intraoperative Tissue Perfusion Measurement by Laser Speckle Imaging: A Potential Aid for Reducing Postoperative Complications in Free Flap Breast Reconstruction.

Adequate tissue perfusion is essential to minimize postoperative complications following microsurgery. Intraoperative knowledge of tissue perfusion could aid surgical decision-making and result in reduced complications. Laser speckle imaging is a new, noninvasive technique for mapping tissue perfusion. This article discusses the feasibility of using laser speckle imaging during free flap breast reconstruction and its potential to identify areas of inadequate perfusion, thus reducing surgical complications. Adult patients scheduled to undergo free flap breast reconstruction were recruited into the study. Laser speckle images were obtained from the abdominal and breast areas at different stages intraoperatively. Zonal perfusion was compared with the Holm classification and clinical observations. Twenty patients scheduled to undergo free flap breast reconstruction were recruited (23 reconstructed breasts) (mean age, 50 years; range, 32 to 68 years). Flap zonal perfusion was 238 (187 to 313), 222 (120 to 265), 206 (120 to 265), and 125 (102 to 220) perfusion units for zones I, II, III, and IV, respectively (analysis of variance, p < 0.0001). Zonal area with perfusion below an arbitrary perfusion threshold were 20 (0.3 to 75), 41 (3 to 99), 49 (9 to 97), and 99 (25 to 100) percent, respectively (analysis of variance, p < 0.0001). One example is presented to illustrate potential intraoperative uses for laser speckle imaging. This study shows that laser speckle imaging is a feasible, noninvasive technique for intraoperative mapping of tissue perfusion during free flap breast reconstruction. Zonal tissue perfusion was reduced across the Holm classification. Observations indicated the potential for laser speckle imaging to provide additional information to augment surgical decision-making by detection of inadequate tissue perfusion. This highlights the opportunity for surgeons to consider additional aids for intraoperative tissue perfusion assessment to help reduce perfusion-related complications. CLINICAL QUESTION/LEVEL OF EVIDENCE:: Diagnostic, IV.

Genetic identification of a novel NeuroD1 function in the early differentiation of islet alpha, PP and epsilon cells.

Nkx2.2 and NeuroD1 are vital for proper differentiation of pancreatic islet cell types. Nkx2.2-null mice fail to form beta cells, have reduced numbers of alpha and PP cells and display an increase in ghrelin-producing epsilon cells. NeuroD1-null mice display a reduction of alpha and beta cells after embryonic day (e) 17.5. To begin to determine the relative contributions of Nkx2.2 and NeuroD1 in islet development, we generated Nkx2.2-/-;NeuroD1-/- double knockout (DKO) mice. As expected, the DKO mice fail to form beta cells, similar to the Nkx2.2-null mice, suggesting that the Nkx2.2 phenotype may be dominant over the NeuroD1 phenotype in the beta cells. Surprisingly, however, the alpha, PP and epsilon phenotypes of the Nkx2.2-null mice are partially rescued by the simultaneous elimination of NeuroD1, even at early developmental time points when NeuroD1 null mice alone do not display a phenotype. Our results indicate that Nkx2.2 and NeuroD1 interact to regulate pancreatic islet cell fates, and this epistatic relationship is cell-type dependent. Furthermore, this study reveals a previously unappreciated early function of NeuroD1 in regulating the specification of alpha, PP and epsilon cells.

NeuroD: the predicted and the surprising.

NeuroD (otherwise known as BETA2) is a basic helix-loop-helix (bHLH) transcription factor that is capable of converting embryonic epidermal cells into fully differentiated neurons in Xenopus embryos. In insulinoma cells, NeuroD can bind and activate the insulin promoter. When NeuroD is deleted in mice, the early differentiating pancreatic endocrine cells and a subset of the neurons in the central and peripheral nervous systems die, resulting in cellular deficits in the pancreatic islets, cerebellum, hippocampus and inner ear sensory ganglia. As a consequence, mice become diabetic and display neurological defects including ataxia and deafness. These gain-of-function and loss-of-function phenotypes suggest that NeuroD controls both common and distinct sets of molecules involved in cell survival and differentiation in different tissue types. In this review, we examine what is known about NeuroD and what remains to be answered. Understanding the primary function of NeuroD will be extremely valuable in the diagnosis and cure of the diseases that involve this transcription factor, which plays essential roles in the development and function of the pancreas and the nervous system.

Recovery of neurological functions in non-human primate model of Parkinson's disease by transplantation of encapsulated neonatal porcine choroid plexus cells.

Parkinson's disease (PD) is a neurodegenerative disease that is primarily characterized by degeneration of dopaminergic (DA) neurons in the substantia nigra (SN) and a loss of their fibre projections in the striatum. We utilized the neonatal porcine choroid plexus (CP), an organ that secretes cerebrospinal fluid containing various types of neurotrophic and neuroprotective factors, to ameliorate the Parkinsonian symptoms in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated rhesus monkeys without requiring immunosuppression. We demonstrate that transplanted encapsulated CP clusters (eCPs) significantly improved neurological functions in MPTP-treated monkeys during the course of six months after transplantation (p < 0.001) when compared with monkeys implanted with empty capsules or subjected to sham surgery. The improvement in neurological scores was accompanied by a corresponding improvement in apomorphine-induced circling behaviour (p < 0.001) as well as increased tyrosine hydroxylase (TH) staining in the striatum. Our results suggest that eCPs are a promising cell therapeutic agent to treat Parkinson's disease.

Pancreatic beta cells require NeuroD to achieve and maintain functional maturity.

NeuroD, a transactivator of the insulin gene, is critical for development of the endocrine pancreas, and NeuroD mutations cause MODY6 in humans. To investigate the role of NeuroD in differentiated beta cells, we generated mice in which neuroD is deleted in insulin-expressing cells. These mice exhibit severe glucose intolerance. Islets lacking NeuroD respond poorly to glucose and display a glucose metabolic profile similar to immature beta cells, featuring increased expression of glycolytic genes and LDHA, elevated basal insulin secretion and O2 consumption, and overexpression of NPY. Moreover, the mutant islets appear to have defective K(ATP) channel-mediated insulin secretion. Unexpectedly, virtually all insulin in the mutant mice is derived from ins2, whereas ins1 expression is almost extinguished. Overall, these results indicate that NeuroD is required for beta cell maturation and demonstrate the importance of NeuroD in the acquisition and maintenance of fully functional glucose-responsive beta cells.

Context-dependent regulation of NeuroD activity and protein accumulation.

NeuroD/BETA2 (referred to as NeuroD hereafter) is a basic helix-loop-helix (bHLH) transcription factor that is required for the development and survival of a subset of neurons and pancreatic endocrine cells in mice. Gain-of-function analyses demonstrated that NeuroD can (i) convert epidermal fate into neuronal fate when overexpressed in Xenopus embryos, and (ii) activate the insulin promoter in pancreatic beta cell lines in response to glucose stimulation. In glucose-stimulated INS-1 pancreatic beta cells, mutations of S259, S266, and S274 to alanines inhibited the ability of NeuroD to activate the insulin promoter. Phosphorylation of those serine residues by ERK1/2 was required for NeuroD activity in that assay. To determine whether the same residues are implicated in the neurogenic activity of NeuroD, we mutated the conserved S259, S266, and S274 of Xenopus NeuroD to alanines (S259A, S266A, and S274A), and performed an ectopic neurogenesis assay in Xenopus embryos. In contrast to what has been observed in the pancreatic beta cell line, the S266A and S274A mutant forms of Xenopus NeuroD displayed significantly increased abilities to form ectopic neurons, while S259A had little effect. In addition, S266A and S274A of Xenopus NeuroD resulted in increased accumulation of protein in the injected embryos while the corresponding mutations on mouse NeuroD did not have the same effect in an insulinoma cell line. Our results demonstrate that the consequence of NeuroD protein modification is context-dependent at both the molecular and functional levels.

Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2.

NeuroD (ND) is a basic helix-loop-helix transcription factor important for neuronal development and survival. By using a yeast two-hybrid screen, we identified two proteins that interact with ND, huntingtin-associated protein 1 (HAP1) and mixed-lineage kinase 2 (MLK2), both of which are known to interact with huntingtin (Htt). Htt is a ubiquitous protein important for neuronal transcription, development, and survival, and loss of its function has been implicated in the pathogenesis of Huntington's disease, a neurodegenerative disorder. However, the mechanism by which Htt exerts its neuron-specific function at the molecular level is unknown. Here we report that Htt interacts with ND via HAP1, and that MLK2 phosphorylates and stimulates the activity of ND. Furthermore, we show that Htt and HAP1 facilitate the activation of ND by MLK2. To our knowledge, ND is the first example of a neuron-specific transcription factor involved in neuronal development and survival whose activity is modulated by Htt. We propose that Htt, together with HAP1, may function as a scaffold for the activation of ND by MLK2.

Math3 and NeuroD regulate amacrine cell fate specification in the retina.

The basic helix-loop-helix genes Math3 and NeuroD are expressed by differentiating amacrine cells, retinal interneurons. Previous studies have demonstrated that a normal number of amacrine cells is generated in mice lacking either Math3 or NEUROD: We have found that, in Math3-NeuroD double-mutant retina, amacrine cells are completely missing, while ganglion and Müller glial cells are increased in number. In the double-mutant retina, the cells that would normally differentiate into amacrine cells did not die but adopted the ganglion and glial cell fates. Misexpression studies using the developing retinal explant cultures showed that, although Math3 and NeuroD alone only promoted rod genesis, they significantly increased the population of amacrine cells when the homeobox gene Pax6 or Six3 was co-expressed. These results indicate that Math3 and NeuroD are essential, but not sufficient, for amacrine cell genesis, and that co-expression of the basic helix-loop-helix and homeobox genes is required for specification of the correct neuronal subtype.

Requirement of multiple basic helix-loop-helix genes for retinal neuronal subtype specification.

Retinal precursor cells give rise to six types of neurons and one type of glial cell during development, and this process is controlled by multiple basic helix-loop-helix (bHLH) genes. However, the precise mechanism for specification of retinal neuronal subtypes, particularly horizontal neurons and photoreceptors, remains to be determined. Here, we examined retinas with three different combinations of triple bHLH gene mutations. In retinas lacking the bHLH genes Ngn2, Math3, and NeuroD, horizontal neurons as well as other neurons such as bipolar cells were severely decreased in number. In the retina lacking the bHLH genes Mash1, Ngn2, and Math3, horizontal and other neurons were severely decreased, whereas ganglion cells were increased. In the retina lacking the bHLH genes Mash1, Math3, and NeuroD, photoreceptors were severely decreased, whereas ganglion cells were increased. In all cases, glial cells were increased. The increase and decrease of these cells were the result of cell fate changes and cell death and seem to be partly attributable to the remaining bHLH gene expression, which also changes because of triple bHLH gene mutations. These results indicate that multiple bHLH genes cross-regulate each other, cooperatively specify neuronal subtypes, and regulate neuronal survival in the developing retina.