Immp2l Enhances the Structure and Function of Mitochondrial Gpd2 Dehydrogenase.

'Inner mitochondrial membrane peptidase 2 like' (IMMP2L) is a nuclear-encoded mitochondrial peptidase that has been conserved through evolutionary history, as has its target enzyme, 'mitochondrial glycerol phosphate dehydrogenase 2' (GPD2). IMMP2L is known to cleave the mitochondrial transit peptide from GPD2 and another nuclear-encoded mitochondrial respiratory-related protein, cytochrome C1 (CYC1). However, it is not known whether IMMP2L peptidase activates or alters the activity or respiratory-related functions of GPD2 or CYC1. Previous investigations found compelling evidence of behavioural change in the Immp2lKD-/- KO mouse, and in this study, EchoMRI analysis found that the organs of the Immp2lKD-/- KO mouse were smaller and that the KO mouse had significantly less lean mass and overall body weight compared with wildtype littermates (p < 0.05). Moreover, all organs analysed from the Immp2lKD-/- KO had lower relative levels of mitochondrial reactive oxygen species (mitoROS). The kidneys of the Immp2lKD-/- KO mouse displayed the greatest decrease in mitoROS levels that were over 50% less compared with wildtype litter mates. Mitochondrial respiration was also lowest in the kidney of the Immp2lKD-/- KO mouse compared with other tissues when using succinate as the respiratory substrate, whereas respiration was similar to the wildtype when glutamate was used as the substrate. When glycerol-3-phosphate (G3P) was used as the substrate for Gpd2, we observed ~20% and ~7% respective decreases in respiration in female and male Immp2lKD-/- KO mice over time. Together, these findings indicate that the respiratory-related functions of mGpd2 and Cyc1 have been compromised to different degrees in different tissues and genders of the Immp2lKD-/- KO mouse. Structural analyses using AlphaFold2-Multimer further predicted that the interaction between Cyc1 and mitochondrial-encoded cytochrome b (Cyb) in Complex III had been altered, as had the homodimeric structure of the mGpd2 enzyme within the inner mitochondrial membrane of the Immp2lKD-/- KO mouse. mGpd2 functions as an integral component of the glycerol phosphate shuttle (GPS), which positively regulates both mitochondrial respiration and glycolysis. Interestingly, we found that nonmitochondrial respiration (NMR) was also dramatically lowered in the Immp2lKD-/- KO mouse. Primary mouse embryonic fibroblast (MEF) cell lines derived from the Immp2lKD-/- KO mouse displayed a ~27% decrease in total respiration, comprising a ~50% decrease in NMR and a ~12% decrease in total mitochondrial respiration, where the latter was consistent with the cumulative decreases in substrate-specific mediated mitochondrial respiration reported here. This study is the first to report the role of Immp2l in enhancing Gpd2 structure and function, mitochondrial respiration, nonmitochondrial respiration, organ size and homeostasis.

Antioxidant Behavioural Phenotype in the Immp2l Gene Knock-Out Mouse.

Mitochondrial dysfunction is strongly associated with autism spectrum disorder (ASD) and the Inner mitochondrial membrane protein 2-like (IMMP2L) gene is linked to autism inheritance. However, the biological basis of this linkage is unknown notwithstanding independent reports of oxidative stress in association with both IMMP2L and ASD. To better understand IMMP2L's association with behaviour, we developed the Immp2lKD knockout (KO) mouse model which is devoid of Immp2l peptidase activity. Immp2lKD -/- KO mice do not display any of the core behavioural symptoms of ASD, albeit homozygous Immp2lKD -/- KO mice do display increased auditory stimulus-driven instrumental behaviour and increased amphetamine-induced locomotion. Due to reports of increased ROS and oxidative stress phenotypes in an earlier truncated Immp2l mouse model resulting from an intragenic deletion within Immp2l, we tested whether high doses of the synthetic mitochondrial targeted antioxidant (MitoQ) could reverse or moderate the behavioural changes in Immp2lKD -/- KO mice. To our surprise, we observed that ROS levels were not increased but significantly lowered in our new Immp2lKD -/- KO mice and that these mice had no oxidative stress-associated phenotypes and were fully fertile with no age-related ataxia or neurodegeneration as ascertained using electron microscopy. Furthermore, the antioxidant MitoQ had no effect on the increased amphetamine-induced locomotion of these mice. Together, these findings indicate that the behavioural changes in Immp2lKD -/- KO mice are associated with an antioxidant-like phenotype with lowered and not increased levels of ROS and no oxidative stress-related phenotypes. This suggested that treatments with antioxidants are unlikely to be effective in treating behaviours directly resulting from the loss of Immp2l/IMMP2L activity, while any behavioural deficits that maybe associated with IMMP2L intragenic deletion-associated truncations have yet to be determined.

Transcriptional Interference Regulates the Evolutionary Development of Speech.

The human capacity to speak is fundamental to our advanced intellectual, technological and social development. Yet so very little is known regarding the evolutionary genetics of speech or its relationship with the broader aspects of evolutionary development in primates. In this study, we describe a large family with evolutionary retrograde development of the larynx and wrist. The family presented with severe speech impairment and incremental retrograde elongations of the pisiform in the wrist that limited wrist rotation from 180° to 90° as in primitive primates. To our surprise, we found that a previously unknown primate-specific gene TOSPEAK had been disrupted in the family. TOSPEAK emerged de novo in an ancestor of extant primates across a 540 kb region of the genome with a pre-existing highly conserved long-range laryngeal enhancer for a neighbouring bone morphogenetic protein gene GDF6. We used transgenic mouse modelling to identify two additional GDF6 long-range enhancers within TOSPEAK that regulate GDF6 expression in the wrist. Disruption of TOSPEAK in the affected family blocked the transcription of TOSPEAK across the 3 GDF6 enhancers in association with a reduction in GDF6 expression and retrograde development of the larynx and wrist. Furthermore, we describe how TOSPEAK developed a human-specific promoter through the expansion of a penta-nucleotide direct repeat that first emerged de novo in the promoter of TOSPEAK in gibbon. This repeat subsequently expanded incrementally in higher hominids to form an overlapping series of Sp1/KLF transcription factor consensus binding sites in human that correlated with incremental increases in the promoter strength of TOSPEAK with human having the strongest promoter. Our research indicates a dual evolutionary role for the incremental increases in TOSPEAK transcriptional interference of GDF6 enhancers in the incremental evolutionary development of the wrist and larynx in hominids and the human capacity to speak and their retrogression with the reduction of TOSPEAK transcription in the affected family.

GDF6 Knockdown in a Family with Multiple Synostosis Syndrome and Speech Impairment.

Multiple synostoses syndrome type 4 (SYNS4; MIM 617898) is an autosomal dominant disorder characterized by carpal-tarsal coalition and otosclerosis-associated hearing loss. SYSN4 has been associated with GDF6 gain-of-function mutations. Here we report a five-generation SYNS4 family with a reduction in GDF6 expression resulting from a chromosomal breakpoint 3' of GDF6. A 30-year medical history of the family indicated bilateral carpal-tarsal coalition in ~50% of affected family members and acquired otosclerosis-associated hearing loss in females only, whereas vertebral fusion was present in all affected family members, most of whom were speech impaired. All vertebral fusions were acquired postnatally in progressive fashion from a very early age. Thinning across the 2nd cervical vertebral interspace (C2-3) in the proband during infancy progressed to block fusion across C2-7 and T3-7 later in life. Carpal-tarsal coalition and pisiform expansion were bilaterally symmetrical within, but varied greatly between, affected family members. This is the first report of SYNS4 in a family with reduced GDF6 expression indicating a prenatal role for GDF6 in regulating development of the joints of the carpals and tarsals, the pisiform, ears, larynx, mouth and face and an overlapping postnatal role in suppression of aberrant ossification and synostosis of the joints of the inner ear (otosclerosis), larynx and vertebrae. RNAseq gene expression analysis indicated >10 fold knockdown of NOMO3, RBMXL1 and NEIL2 in both primary fibroblast cultures and fresh white blood cells. Together these results provide greater insight into the role of GDF6 in skeletal joint development.

siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference.

Exogenous siRNAs are commonly used to regulate endogenous gene expression levels for gene function analysis, genotype-phenotype association studies and for gene therapy. Exogenous siRNAs can target mRNAs within the cytosol as well as nascent RNA transcripts within the nucleus, thus complicating siRNA targeting specificity. To highlight challenges in achieving siRNA target specificity, we targeted an overlapping gene set that we found associated with a familial form of multiple synostosis syndrome type 4 (SYSN4). In the affected family, we found that a previously unknown non-coding gene TOSPEAK/C8orf37AS1 was disrupted and the adjacent gene GDF6 was downregulated. Moreover, a conserved long-range enhancer for GDF6 was found located within TOSPEAK which in turn overlapped another gene which we named SMALLTALK/C8orf37. In fibroblast cell lines, SMALLTALK is transcribed at much higher levels in the opposite (convergent) direction to TOSPEAK. siRNA targeting of SMALLTALK resulted in post transcriptional gene silencing (PTGS/RNAi) of SMALLTALK that peaked at 72 h together with a rapid early increase in the level of both TOSPEAK and GDF6 that peaked and waned after 24 h. These findings indicated the following sequence of events: Firstly, the siRNA designed to target SMALLTALK mRNA for RNAi in the cytosol had also caused an early and transient transcriptional interference of SMALLTALK in the nucleus; Secondly, the resulting interference of SMALLTALK transcription increased the transcription of TOSPEAK; Thirdly, the increased transcription of TOSPEAK increased the transcription of GDF6. These findings have implications for the design and application of RNA and DNA targeting technologies including siRNA and CRISPR. For example, we used siRNA targeting of SMALLTALK to successfully restore GDF6 levels in the gene therapy of SYNS4 family fibroblasts in culture. To confidently apply gene targeting technologies, it is important to first determine the transcriptional interference effects of the targeting reagent and the targeted gene.

LRRTM4 Terminal Exon Duplicated in Family with Tourette Syndrome, Autism and ADHD.

Tourette syndrome (TS) is a neurodevelopmental disorder characterised by motor and vocal tics and strong association with autistic deficits, obsessive-compulsive disorder (OCD) and attention-deficit/hyperactivity disorder (ADHD). The genetic overlap between TS and autism spectrum disorder (ASD) includes those genes that encode the neurexin trans-synaptic connexus (NTSC) inclusive of the presynaptic neurexins (NRXNs) and postsynaptic neuroligins (NLGNs), cerebellin precursors (CBLNs in complex with the glutamate ionotropic receptor deltas (GRIDs)) and the leucine-rich repeat transmembrane proteins (LRRTMs). In this study, we report the first evidence of a TS and ASD association with yet another NTSC gene family member, namely LRRTM4. Duplication of the terminal exon of LRRTM4 was found in two females with TS from the same family (mother and daughter) in association with autistic traits and ASD.

Tourette Syndrome Risk Genes Regulate Mitochondrial Dynamics, Structure, and Function.

Gilles de la Tourette syndrome (GTS) is a neurodevelopmental disorder characterized by motor and vocal tics with an estimated prevalence of 1% in children and adolescents. GTS has high rates of inheritance with many rare mutations identified. Apart from the role of the neurexin trans-synaptic connexus (NTSC) little has been confirmed regarding the molecular basis of GTS. The NTSC pathway regulates neuronal circuitry development, synaptic connectivity and neurotransmission. In this study we integrate GTS mutations into mitochondrial pathways that also regulate neuronal circuitry development, synaptic connectivity and neurotransmission. Many deleterious mutations in GTS occur in genes with complementary and consecutive roles in mitochondrial dynamics, structure and function (MDSF) pathways. These genes include those involved in mitochondrial transport (NDE1, DISC1, OPA1), mitochondrial fusion (OPA1), fission (ADCY2, DGKB, AMPK/PKA, RCAN1, PKC), mitochondrial metabolic and bio-energetic optimization (IMMP2L, MPV17, MRPL3, MRPL44). This study is the first to develop and describe an integrated mitochondrial pathway in the pathogenesis of GTS. The evidence from this study and our earlier modeling of GTS molecular pathways provides compounding support for a GTS deficit in mitochondrial supply affecting neurotransmission.

Secondary association of PDLIM5 with paranoid schizophrenia in Emirati patients.

Schizophrenia is a clinically and genetically heterogeneous disorder of unknown etiology. PDLIM5 variants have been linked to schizophrenia and other related neuropsychiatric disorders and upregulated in the brain of schizophrenia patients suggesting a possible pathogenic role in disease progression. The aim of this study is to examine the potential association of schizophrenia in Emirati patients with previously reported variants in PDLIM5, PICK1, NRG3 or DISC1 genes. Consequently, we found a secondary association between PDLIM5 variants and the paranoid subtype of schizophrenia in Emirati Arabs suggesting that PDLIM5 may represent a determinate/marker for schizophrenia subtype specification. However, no associations were found with variants in PICK1, NRG3 or DISC1 genes.

Targeted alpha anticancer therapies: update and future prospects.

Targeted alpha therapy (TAT) is an emerging option for local and systemic cancer treatment. Preclinical research and clinical trials show that alpha-emitting radionuclides can kill targeted cancer cells while sparing normal cells, thus reducing toxicity. (223)RaCl2 (Xofigo(®)) is the first alpha emitting radioisotope to gain registration in the US for palliative therapy of prostate cancer bone metastases by indirect physiological targeting. The alpha emitting radioisotopes (211)At, (213)Bi, (225)Ac and (227)Th are being used to label targeting vectors such as monoclonal antibodies for specific cancer therapy indications. In this review, safety and tolerance aspects are considered with respect to microdosimetry, specific energy, Monte Carlo model calculations, biodosimetry, equivalent dose and mutagenesis. The clinical efficacy of TAT for solid tumors may also be enhanced by its capacity for tumor anti-vascular (TAVAT) effects. This review emphasizes key aspects of TAT research with respect to the PAI2-uPAR complex and the monoclonal antibodies bevacizumab, C595 and J591. Clinical trial outcomes are reviewed for neuroendocrine tumors, leukemia, glioma, melanoma, non-Hodgkins lymphoma, and prostate bone metastases. Recommendations and future directions are proposed.

Balance within the Neurexin Trans-Synaptic Connexus Stabilizes Behavioral Control.

Autism spectrum disorder (ASD) is characterized by a broad spectrum of behavioral deficits of unknown etiology. ASD associated mutations implicate numerous neurological pathways including a common association with the neurexin trans-synaptic connexus (NTSC) which regulates neuronal cell-adhesion, neuronal circuitry, and neurotransmission. Comparable DNA lesions affecting the NTSC, however, associate with a diversity of behavioral deficits within and without the autism spectrum including a very strong association with Tourette syndrome. The NTSC is comprised of numerous post-synaptic ligands competing for trans-synaptic connection with one of the many different neurexin receptors yet no apparent association exists between specific NTSC molecules/complexes and specific behavioral deficits. Together these findings indicate a fundamental role for NTSC-balance in stabilizing pre-behavioral control. Further molecular and clinical characterization and stratification of ASD and TS on the basis of NTSC status will help elucidate the molecular basis of behavior - and define how the NTSC functions in combination with other molecular determinates to strengthen behavioral control and specify behavioral deficits.

Next-generation therapy for residual prostate cancer.

Prostate cancer claimed an estimated 136,500 lives globally in 2011. Ironically, the best existing treatment strategies provide survival benefits and missed opportunities to further improve outcomes. Prostatectomy provides the greatest survival benefit, albeit the risk of systemic recurrence increases dramatically with extracapsular involvement. To date, further systemic treatment is not generally prescribed for these 'high-risk' patients until such time as advanced disease is diagnosed based on persistent high PSA levels and/or when larger tumors are confirmed by imaging. This recurrent form of the disease is most often terminal. Androgen deprivation therapy (ADT) provides outstanding early control for these patients, which is rather tragic as the early benefits of ADT are lost within 2 years for most men, as the cancer again progresses to an incurable 'late-stage' castration-resistant form of the disease with a median survival of approximately 18 months. We review the potential of targeted α-therapy as an adjuvant with minimal side effects for early-stage high-risk patients to be administered immediately following prostatectomy and/or during ADT.

Smg1 haploinsufficiency predisposes to tumor formation and inflammation.

SMG1 is a member of the phosphoinositide kinase-like kinase family of proteins that includes ATM, ATR, and DNA-PK, proteins with known roles in DNA damage and cellular stress responses. SMG1 has a well-characterized role in nonsense-mediated decay as well as suggested roles in the DNA damage response, resistance to oxidative stress, regulation of hypoxic responses, and apoptosis. To understand the roles of SMG1 further, we generated a Genetrap Smg1 mouse model. Smg1 homozygous KO mice were early embryonic lethal, but Smg1 heterozygous mice showed a predisposition to a range of cancers, particularly lung and hematopoietic malignancies, as well as development of chronic inflammation. These mice did not display deficiencies in known roles of SMG1, including nonsense-mediated decay. However, they showed elevated basal tissue and serum cytokine levels, indicating low-level inflammation before the development of tumors. Smg1 heterozygous mice also showed evidence of oxidative damage in tissues. These data suggest that the inflammation observed in Smg1 haploinsufficiency contributes to susceptibility to cancer and that Smg1-deficient animals represent a model of inflammation-enhanced cancer development.

The senescence pathway in prostatic carcinogenesis.

Prostate cancer is a disease of the old and with increasing life expectancy, its incidence will continue to increase in the future. Control of prostate cancer has involved androgen ablation as a routine form of therapy. However, after an initial response, therapy-resistant clones can appear and result in cancer progression and metastasis with high mortality. The precise mechanisms for the development of androgen resistance are yet uncertain. It appears to be multi-factorial and relates not only to newly acquired genomic capabilities of the cancer cells but also to their interaction with their microenvironment. Overcoming cellular senescence is essential for oncogenesis. Although it seems to be a protective response for normal cells to avoid malignant transformation, senescence can on the other hand promote tumour progression. Interaction of senescent cancer cells with their microenvironment may be the key link to survival or regression of neoplastic cells. Hence, there is speculation that senescence may be a useful new target for therapy in the future. We review the role of senescence in prostate cancer and the effect of tumour microenvironment on androgen resistance.

Low levels of ATM in breast cancer patients with clinical radiosensitivity.

BACKGROUND AND PURPOSE: Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues. In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity. The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM protein. RESULTS: Semi-quantitative immuno-blotting provided a reliable and reproducible method to compare levels of the ATM protein for a rare cohort of 20 cancer patients selected on the basis of their severe adverse normal tissue reactions to radiotherapy. We found that 4/12 (33%) of the breast cancer patients with severe adverse normal tissue reactions following radiotherapy had ATM protein levels < 55% compared to the mean for non-reactor controls. CONCLUSIONS: ATM mutations are generally considered low risk alleles for breast cancer and clinical radiosensitivity. From results reported here we propose a tentative ATM protein threshold of ~55% for high-risk of clinical radiosensitivity for breast cancer patients.

Markers for detection of prostate cancer.

Early detection of prostate cancer is problematic, not just because of uncertainly whether a diagnosis will benefit an individual patient, but also as a result of the imprecise and invasive nature of establishing a diagnosis by biopsy. Despite its low sensitivity and specificity for identifying patients harbouring prostate cancer, serum prostate specific antigen (PSA) has become established as the most reliable and widely-used diagnostic marker for this condition. In its wake, many other markers have been described and evaluated. This review focuses on the supporting evidence for the most prominent of these for detection and also for predicting outcome in prostate cancer.

New genomic structure for prostate cancer specific gene PCA3 within BMCC1: implications for prostate cancer detection and progression.

BACKGROUND: The prostate cancer antigen 3 (PCA3/DD3) gene is a highly specific biomarker upregulated in prostate cancer (PCa). In order to understand the importance of PCA3 in PCa we investigated the organization and evolution of the PCA3 gene locus. METHODS/PRINCIPAL FINDINGS: We have employed cDNA synthesis, RTPCR and DNA sequencing to identify 4 new transcription start sites, 4 polyadenylation sites and 2 new differentially spliced exons in an extended form of PCA3. Primers designed from these novel PCA3 exons greatly improve RT-PCR based discrimination between PCa, PCa metastases and BPH specimens. Comparative genomic analyses demonstrated that PCA3 has only recently evolved in an anti-sense orientation within a second gene, BMCC1/PRUNE2. BMCC1 has been shown previously to interact with RhoA and RhoC, determinants of cellular transformation and metastasis, respectively. Using RT-PCR we demonstrated that the longer BMCC1-1 isoform - like PCA3 - is upregulated in PCa tissues and metastases and in PCa cell lines. Furthermore PCA3 and BMCC1-1 levels are responsive to dihydrotestosterone treatment. CONCLUSIONS/SIGNIFICANCE: Upregulation of two new PCA3 isoforms in PCa tissues improves discrimination between PCa and BPH. The functional relevance of this specificity is now of particular interest given PCA3's overlapping association with a second gene BMCC1, a regulator of Rho signalling. Upregulation of PCA3 and BMCC1 in PCa has potential for improved diagnosis.

Tourette syndrome and klippel-feil anomaly in a child with chromosome 22q11 duplication.

This is the first case description of the association of Klippel-Feil Syndrome (KFS), Tourette Syndrome (TS), Motor Stereotypies, and Obsessive Compulsive Behavior, with chromosome 22q11.2 Duplication Syndrome (22q11DupS). Neuropsychiatric symptoms in persons with 22q11.2 deletion, including obsessive compulsiveness, anxiety, hyperactivity, and one prior case report of TS, have been attributed to low copy number effects on Catechol-O-Methyltransferase (COMT). However, the present unique case of 22q11DupS and TS suggests a more complex relationship, either for low- or high-COMT activity, or for other genes at this locus.

Mutations in GDF6 are associated with vertebral segmentation defects in Klippel-Feil syndrome.

Klippel-Feil syndrome (KFS) is a congenital disorder of spinal segmentation distinguished by the bony fusion of anterior/cervical vertebrae. Scoliosis, mirror movements, otolaryngological, kidney, ocular, cranial, limb, and/or digit anomalies are often associated. Here we report mutations at the GDF6 gene locus in familial and sporadic cases of KFS including the recurrent missense mutation of an extremely conserved residue c.866T>C (p.Leu289Pro) in association with mirror movements and an inversion breakpoint downstream of the gene in association with carpal, tarsal, and vertebral fusions. GDF6 is expressed at the boundaries of the developing carpals, tarsals, and vertebrae and within the adult vertebral disc. GDF6 knockout mice are best distinguished by fusion of carpals and tarsals and GDF6 knockdown in Xenopus results in a high incidence of anterior axial defects consistent with a role for GDF6 in the etiology, diversity, and variability of KFS.

Multiple sclerosis in a radiosensitive family with low levels of the ATM protein.

Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system (CNS) characterized by demyelination associated with progressive disability. The mechanisms underlying the pathogenesis of MS remain a mystery. The highly pleiotropic syndrome known as ataxia telangiectasia (A-T) overlaps with MS in that it also presents with demyelination in the CNS. Whether demyelination in MS or in A-T is initiated through neuronal degeneration or immune dysfunction is not yet known. However, unlike MS, the underlying cause of A-T is known to result from mutations in the A-T gene (ATM) that often result in the complete loss of ATM protein and loss/gain of function. ATM is implicated in neurological degeneration, particularly in the cerebellum, cellular apoptosis, immunodeficiency, double stranded deoxyribonucleic acid (DNA) rejoining, VDJ antibody recombination, tumour suppression, particularly T-lymphoid malignancies, signal transduction, cell-cycle control and cellular radiohypersensitivity. In this study, we describe a case of MS in a family with cellular radiosensitivity and abnormally low postinduction levels of the ATM protein. Defective DNA repair/rejoining may impact on autoimmunity.