High doses of 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") are known to reduce levels of various serotonergic markers outside of the raphe nuclei. To test the hypothesis that these deficits reflect a degeneration of distal axons/terminals, we investigated the effects of an MDMA binge (10mg/kg x 4) on the relative protein and genetic expression of several serotonergic markers in rats, as well as the effects of this compound on the quantity of serotonergic terminals in these animals. In experiment I, we examined whether MDMA alters serotonin transporter (SERT) levels as determined by lysate binding and immunoblotting analyses. Both methods of analysis revealed MDMA-induced reductions in regional SERT content. Experiment II investigated MDMA-induced changes in terminal-specific levels of SERT and the vesicular monoamine transporter 2 (VMAT-2) in the hippocampus, a region with sparse dopaminergic innervation, after lesioning noradrenergic input with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). Animals were administered 100 mg/kg DSP-4 or saline 1 week prior to MDMA (or saline). As determined by immunoblotting of synaptosomal tissue, the DSP-4/MDMA group showed little change in hippocampal VMAT-2 protein expression compared to DSP-4/Saline controls, despite large reductions in SERT levels in all regions examined in the MDMA-treated animals. Experiment III examined whether MDMA alters genetic expression of SERT and VMAT-2. When compared to saline-treated controls, animals given MDMA showed a striking decrease in SERT gene expression (and a lesser effect on VMAT-2) in dorsal/median raphe as assessed by quantitative RT-PCR. Experiment IV(a) investigated the effects of MDMA on gene and protein expression of tryptophan hydroxylase (TPH) in the hippocampus. Levels of TPH protein were unchanged between treatment groups, while transcript levels were decreased 15-fold in the dorsal/median raphe. In experiment IV(b), flow cytometry was used to measure whether MDMA alters the quantity of serotonergic terminals in the hippocampus. MDMA-treated animals showed an increase in the number of serotonergic synaptosomes identified by co-labeling for synaptosome-associated protein of 25 kDa (SNAP-25) and TPH. These results demonstrate that MDMA causes substantial regulatory changes in the expression of serotonergic markers with no evidence for synaptic loss, questioning the need to invoke distal axotomy as an explanation of MDMA-related serotonergic deficits.

Neurotrophins, such as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), are key factors in neuronal survival and function. In Alzheimer's disease (AD), there is a change in the normal level of these neurotrophins and their precursors (proNGF and proBDNF). The mechanism/s underlying changes in the levels of these neurotrophins in AD is not fully understood. According to the amyloid cascade hypothesis, amyloid-β is the original insult in AD and tau pathology is a downstream event. Amyloid-β interferes with axonal transport and reduces BDNF levels. However, it is not clear if amyloid-β affects neurotrophin levels directly or through tau hyperphosphorylation. If tau is responsible for changes in the level of neurotrophins in AD, we expect to observe the same alteration in neurotrophin levels in other diseases with tau dysfunction such as tauopathies. We tested the levels of BDNF mRNA and proNGF protein in subjects with tauopathies. We observed significant decrease in BDNF mRNA levels in subjects with Corticobasal degeneration. Our result suggests that BDNF may be down-regulated by tau hyperphosphorylation. Moreover, we showed that there was a significant increase in the level of proNGF in Pick's disease (PiD). Interestingly, AD and PiD share common tau modifications. Our result demonstrates a role for tau dysfunction in changes in the level of proNGF. Therefore, study of the levels of NGF and BDNF in non-AD tauopathies has shed light on the mechanisms underlying neurotrophin dysregulation in AD. How do increased levels of proNGF impact the brain in AD or PiD? Is neuronal degeneration in AD or PiD due to the lack of neurotrophic support of proNGF or do increased levels of apoptotic proNGF cause neurodegeneration? Lee et al. (2001b) and Fahnestock et al. (2004a) produced two different cleavage-resistant proNGFs with opposite activities (apoptotic versus neurotrophic). Structural and procedural differences between the two cleavage resistant proNGFs and different bioassays can cause opposite activities. We showed that proNGF from Lee's lab was neurotrophic when it was expressed in the expression system used by Fahnestock et al. or when it was purified. ProNGF expressed in a different expression system was also neurotrophic. ProNGF was neurotrophic in all bioassays except the serum withdrawal assay. We conclude that proNGF is normally neurotrophic but may be apoptotic when cell survival is already compromised. We propose that in AD, cells undergo degeneration due to the lack of neurotrophic support of proNGF (impaired transport). Moreover, TrkA is downregulated in AD which compromises cell survival and may lead to apoptosis induced by increased levels of proNGF. / Thesis / Doctor of Philosophy (PhD)

Parkinson’s disease (PD) has long been conceptualized as a motor disorder, but significant clinical features arise before motor symptoms are present. Although prospective, longitudinal research offers the most valid approach for determining pre-diagnostic indicators of PD, it is costly and requires a long time-course. Leveraging existing epidemiological datasets offers the opportunity to identify pre-diagnostic features that may predict later PD diagnosis. This project used the Framingham Heart Study (FHS) database of prospective follow-up on a community-based sample that spans over six decades. Regular surveillance identified 156 incident cases of PD. Promising biomarker and other clinical marker candidates were derived from cohort-based samples without prospective follow-up and included cognition, depression, and inflammation. The main hypothesis was that potential markers would discriminate between individuals who did/ did not go on to a later PD diagnosis. The FHS database provided clinical markers (cognition, depression) and fluid biomarkers (levels of plasma inflammation) for interrogation. Cognition was indexed by performance on the Mini-Mental State Examination and a comprehensive neuropsychological assessment, including measures of attention, memory, and executive functioning. Depression was derived from scores on the Center for Epidemiologic Studies Depression Scale (CES-D). Separate means comparison and logistic regression analyses to maximize sample sizes were conducted on available data for candidate (bio)markers at time-points 1-3 years or 1-5 years pre-diagnosis for PD cases (N=7-33) and control participants (N=28-224), in samples matched for age, sex, and education level. No significant differences were found between PD and control participants on any measure of cognitive functioning 1-3 years pre-diagnosis. No significant differences were found for total CES-D scores or levels of plasma inflammation 1-5 years pre-diagnosis. Higher levels of C-reactive protein and TNF-alpha were significantly correlated with increasing age in the total sample but not for PD specifically. These results indicate that cognition, self-reported depression, and plasma inflammation may not be useful as markers of PD risk, and efforts should likely focus on alternative candidate markers. Detecting PD in the earliest stages is an important goal, as it could lead to treatments that attenuate progression, improve clinical prognosis, and enhance the possibility of disease prevention. / 2021-02-20T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / Glaucoma is a leading cause of blindness characterized by the progressive loss of retinal ganglion cells (RGCs), essentially severing the connection between the eye and the brain. Among many underlying causes of the disease, mutations in the Optineurin (OPTN) gene result in severe RGC neurodegeneration in the absence of elevated intraocular pressure, providing a novel opportunity to study molecular mechanisms that lead to RGC neurodegeneration associated with glaucoma. Efforts of this study establishing a human pluripotent stem cell (hPSC)-derived in vitro disease model by inserting OPTN(E50K) mutation via CRISPR/Cas9 genome editing and investigate the cellular mechanisms of RGC neurodegeneration associated with glaucoma. OPTN(E50K) RGCs revealed neurodegeneration phenotypes, including downregulation of RGCs transcription factors, neurite retraction, and hyperexcitability, suggesting that OPTN(E50K) RGCs can serve as an appropriate disease model to study glaucoma-associated neurodegeneration. Since OPTN serves a primary role as an autophagy receptor, we further hypothesized that the OPTN(E50K) mutation disrupts autophagy in RGCs, and modulation of autophagy by mammalian target of rapamycin (mTOR)-independent pathways can preserve RGC phenotypes by maintaining mTOR signaling. OPTN(E50K) RGCs exhibited a higher number of OPTN puncta along with an overall reduced expression of OPTN protein, indicating a gain of toxic protein accumulation or loss of protein function. Furthermore, OPTN(E50K) RGCs revealed an accumulation of the autophagosome protein LC3 in a punctal manner as well as increased expression of lysosomal proteins, suggesting a disruption of degradation pathway in autophagosome and lysosome fusion. As mTOR complex 1 (mTORC1) signaling serves as a negative regulator of autophagy, a downregulation of mTORC1 signaling via activation of stress sensor adenosine monophosphate-activated protein kinase (AMPK) was observed as a possible compensatory mechanism for autophagy deficits in OPTN(E50K) RGCs. Pharmacological inhibition of mTOR in wild-type hRGCs resulted in similar disease-related phenotypes, while preservation of the mTOR pathway in OPTN(E50K) RGCs by treatment with the mTOR-independent autophagy modulator trehalose cleared OPTN accumulated puncta, preserving mTORC1 signaling, as well as rescuing neurodegenerative phenotypes. To further validate these associations in an animal model, the microbead occlusion mouse model was established by injection of magnetic microbeads in the anterior chamber to block aqueous outflow resulting ocular hypertension. In agreement with our findings in hRGCs, a decrease in mTOR signaling associated with an increase in the expression of autophagy-associated proteins was observed in RGCs in the microbead occlusion model. Additionally, these disease-related phenotypes were observed specifically within RGCs but not cortical neurons with an underlying OPTN(E50K) mutation, demonstrating that autophagy represents an essential pathway in RGCs to maintain homeostasis, and selective disrupt of autophagy in RGCs leads to neurodegeneration. Taken together, the results of this study highlight an essential balance between autophagy and mTORC1 signaling that is essential for the homeostasis of RGCs, while disruption to these signaling pathways contributes to neurodegenerative features in glaucoma. These results also demonstrated the ability to pharmacologically intervene to experimentally manipulate these pathways and rescue neurodegenerative phenotypes, providing a potential therapeutic target to prevent glaucoma-associated neurodegeneration.

The hexanucleotide repeat expansion GGGGCCn (also known as G4C2n) localizes in the first intron of the C9ORF72 gene and is the most common genetic cause of ALS and FTD (C9ALS/FTD). The pathomechanisms proposed for C9ALS/FTD suggest that from sense (G4C2)n- and anti-sense (C4G2)n-containing transcripts originate two different mechanisms of toxicity: i) by the alteration of RNA processing due to binding and sequestration of RNA-binding proteins, thereby leading to impairment of RNA metabolism; and ii) by their unconventional Repeat-associated non AUG (RAN) translation into five different dipeptide-repeats (DPRs). In addition, pathological expansion of (G4C2)n reduces the C9orf72 transcription causing loss of function of the C9ORF72 protein. The toxicity of some of these DPRs has been showed in several cell lines, in iPSC-derived neurons, in Drosophila and in mouse models. An impairment of the ubiquitin-proteasome system (UPS) due to aggregation of toxic proteins is largely demonstrated in neurodegenerative disorders and among the mechanisms of DPR-related toxicity. RAN translation of (G4C2)n-RNAs has been recently shown to require a near-cognate start codon upstream of the repeat in frame +1 and to be triggered by stress conditions in a cap-dependent or cap-independent way. However, the mechanism regulating RAN translation is still largely unknown. Importantly, no small molecules are known to selectively modulate RAN translation, even if antisense oligonucleotides (ASOs) and small molecules binding the r(GGGGCC)n have been proposed as therapeutics for C9ALS/FTD. In addition, no effective pharmacological approach to reduce the pathological load of DPRs is currently available. Here, I developed a high-throughput drug-screening assay to identify small molecules and relative molecular targets that can modulate the DPR level. Among the identified hits, two hits reduced DPRs expression levels triggering the protein clearance system in vitro. Moreover, the screening identified compounds having the same target that increased DPRs expression levels indicating the targeted pathway as a crucial modulator of the translation process of the C9orf72 repeat-containing mRNAs. Conversely, I showed that pharmacological inhibition of the pathway reduced DPRs expression levels in vitro, while in vivo it rescued climbing ability and increased life span of Drosophila flies carrying G4C2X36 repeats. Moreover, genetic ablation of the target reduced DPRs expression levels by decreasing their translation efficiency in vitro and rescued the pathological phenotype in vivo. Together, the results show the identification of new pathways as new drug targets for C9ALS/FTD.

In this two-part study, I investigated whether the cyclic-adenosine monophosphate response element-binding (CREB) protein has the potential to be clinically modulated as a therapeutic target for the treatment of neurodegenerative diseases. Part one consisted of a systematic review that was conducted on select articles gathered through a stepwise method to explore (1) the relationship between diseased, neurodegenerative brains and levels of active, phosphorylated CREB (pCREB), (2) increased activation of CREB as a treatment for neurodegenerative symptoms, and (3) a potential therapeutic drug for neurodegenerative diseases that can target CREB signaling. The results of the systematic review showed evidence that suggested excitotoxic concentrations of N-methyl-D-aspartate (NMDA) results in decreased pCREB levels, while decreased pCREB levels were associated with impaired cognition and behavior, increased cell death, as well as decreased CRE-gene transcription and long-term potentiation (LTP). Part two consisted of a systematic review and meta-analysis on clinical trials that used the phosphodiesterase type IV inhibitor, roflumilast, on healthy and schizophrenic patients. It was found that 100 µM roflumilast was able to improve verbal learning in healthy and schizophrenic subjects (ES = 64). Initial evidence indicates that future research on neurodegenerative diseases should further investigate CREB’s potential to be clinically modulated and research investigating PDE4 inhibitor drug therapy for the treatment of neurodegeneration should be expanded upon further in subsequent studies.

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