Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery.

Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties

Mechanistic Insights Into MicroRNA-Induced Neuronal Reprogramming of Human Adult Fibroblasts

The use of transcriptional factors as cell fate regulators are often the primary focus in the direct reprogramming of somatic cells into neurons. However, in human adult fibroblasts, deriving functionally mature neurons with high efficiency requires additional neurogenic factors such as microRNAs (miRNAs) to evoke a neuronal state permissive to transcription factors to exert their reprogramming activities. As such, increasing evidence suggests brain-enriched miRNAs, miR-9/9∗ and miR-124, as potent neurogenic molecules through simultaneously targeting of anti-neurogenic effectors while allowing additional transcription factors to generate specific subtypes of human neurons. In this review, we will focus on methods that utilize neuronal miRNAs and provide mechanistic insights by which neuronal miRNAs, in synergism with brain-region specific transcription factors, drive the conversion of human fibroblasts into clinically relevant subtypes of neurons. Furthermore, we will provide insights into the age signature of directly converted neurons and how the converted human neurons can be utilized to model late-onset neurodegenerative disorders

Delineating the Role of MiR-124 for the Activation of Neuronal Program

The ectopic expression of two brain-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), can robustly and efficiently reprogram human skin fibroblasts into neurons. The miRNAs act as repressors of non-neuronal genes in fibroblasts for the induction of the neuronal program. This process is analogous to neurogenesis in vivo when the expression of miR-9/9* and miR-124 represses anti-neurogenic genes such as REST or NRSF (neuron-restrictive silencer factor/repressor element-1 silencing transcription factor). Although we have some mechanistic insights into how miR-9/9*-124 drives fate conversion by acting as negative regulators of gene expression, little remained understood of the role of miRNAs as positive regulators of gene expression for the activation of neuronal fate. In this thesis, we present our current understanding of how miR-9/9*-124 drives neuronal reprogramming as well as the mechanistic insights into how miR-9/9*-124 can promote expression of neuronal genes. Based on Argonuate (AGO) HITS-CLIP, we uncovered that AGO is bound to neuronal transcripts that are progressively upregulated during reprogramming. Such observation suggests that contrary to the canonical repressive roles of miRNAs, miR-9/9*-124 may be playing a positive role in the expression of bound neuronal transcripts. Using PTB family of RNA-binding proteins as an example, we delineate a mechanism by which miR-124 can simultaneously repress PTBP1, the non-neuronal PTB homolog, while promoting the upregulation of PTBP2, the neuronal PTB homolog, in neurons. This process requires the synergism of miRNA targeting as well as a family of neuronal ELAVL proteins (nELAVLs). We further showed that this miRNA- and nELAVL-mediated upregulation of PTBP2 is neither unique to the conversion process nor PTBP2 transcript alone, but also in primary human neurons and is likely a mechanism used to regulate other neuronal transcripts. With PTBP2 expression induced in neurons, PTBP2 is involved in the alternative splicing of numerous neuronal transcripts. Such transcript includes a subunit of the chromatin remodeling complex, DPF1. Although detailed function of DPF1 remains unclear in neurons, different PTBP2-mediated DPF1 spliced isoforms are likely involved in the interaction of different modified histones for the establishment of a neuronal chromatin landscape

Defining functional classes of Barth syndrome mutation in humans

The X-linked disease Barth syndrome (BTHS) is caused by mutations in TAZ; TAZ is the main determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, cardiolipin. To date, a detailed characterization of endogenous TAZ has only been performed in yeast. Further, why a given BTHS-associated missense mutation impairs TAZ function has only been determined in a yeast model of this human disease. Presently, the detailed characterization of yeast tafazzin harboring individual BTHS mutations at evolutionarily conserved residues has identified seven distinct loss-of-function mechanisms caused by patient-associated missense alleles. However, whether the biochemical consequences associated with individual mutations also occur in the context of human TAZ in a validated mammalian model has not been demonstrated. Here, utilizing newly established monoclonal antibodies capable of detecting endogenous TAZ, we demonstrate that mammalian TAZ, like its yeast counterpart, is localized to the mitochondrion where it adopts an extremely protease-resistant fold, associates non-integrally with intermembrane space-facing membranes and assembles in a range of complexes. Even though multiple isoforms are expressed at the mRNA level, only a single polypeptide that co-migrates with the human isoform lacking exon 5 is expressed in human skin fibroblasts, HEK293 cells, and murine heart and liver mitochondria. Finally, using a new genome-edited mammalian BTHS cell culture model, we demonstrate that the loss-of-function mechanisms for two BTHS alleles that represent two of the seven functional classes of BTHS mutation as originally defined in yeast, are the same when modeled in human TAZ

EFFECTS OF FUNCTIONAL KNEE BRACES ON NEUROMUSCULAR ADAPTATION IN ANTERIOR CRUCIATE LIGAMENT INJURED PATIENTS

INTRODUCTION: Neuromuscular adaptation in the lower extremity has been found in anterior cruciate ligament (ACL) injured patients, with mechanical and electromyographic alterations such as reduced knee extensor moment and power, increased hamsrings muscle activity, and decreased muscle strength (Berchuck, Andriacchi, Bach, & Reider, 1990; DeVita, Lassiter, Hortobagyi, & Torry, 1998). Functional knee bracing has been a common method to enhance functional knee stability in these patients for the past three decades. However, the long-term effects of knee bracing on ACL-reconstructed (ACL-R) patients have not been reported in the literature. There is thus an urgent need to identify the effects of bracing on the gait displayed by these patients