Friedreich's Ataxia (FA) is an autosomal-recessive, neurodegenerative disease characterized by progressive lower extremity muscle weakness and sensory loss, balance deficits, limb and gait ataxia, and dysarthria. FA is considered a sensory ataxia because the dorsal root ganglia and spinal cord dorsal columns are involved early in the disease, whereas the cerebellum is affected later. Balance deficits and gait ataxia are often evaluated clinically and in research using clinical rating scales. Recently, quantitative tools such as the Biodex Balance System SD and the GAITRite Walkway System have become available to objectively assess balance and gait, respectively. However, there are limited studies using instrumented measures to quantitatively assess and characterize balance and gait disturbances in FA, and longitudinal, quantitative analyses of both balance and gait have not been investigated in this patient cohort. The purpose of the present study was to characterize gait patterns of adults with FA and to identify changes in gait and balance over time using clinical rating scales and quantitative measures. Additionally, this study investigated the relationship between disease duration, clinical rating scale scores and objective measures of gait and balance. This study used a longitudinal research design to investigate changes in balance and gait in 8 adults with genetically confirmed FA and 8 healthy controls matched for gender, age, height, and weight. Subjects with FA were evaluated using the Berg Balance Scale (BBS), the Friedreich's Ataxia Rating Scale (FARS) and instrumented gait and balance measures at baseline, 6 months, 12 months and 24 months. Controls underwent the same tests at baseline and 12 months. Gait parameters were measured utilizing the GAITRite Walkway system with a focus on gait velocity, cadence, step and stride lengths, step and stride length variability and percent of the gait cycle in swing, stance and double limb support. Balance was assessed using the BBS and the Biodex Balance System; the latter included tests of postural stability and limits of stability. At baseline, there were significant differences in gait and balance parameters, BBS scores and FARS total scores between FA subjects and controls as determined using paired t-tests (p This is the first longitudinal study to demonstrate changes over time in gait and balance of adults with FA using both quantitative measures and clinical rating scales. This study provided a detailed characterization of the gait pattern and balance of adults with FA. The GAITRite Walkway system proved to be a sensitive measure, and able to detect subtle changes in gait parameters over time in adults with FA. In addition, the BBS was an appropriate and sensitive assessment to detect changes in static and dynamic balance in this patient cohort. Finally, results revealed a strong and consistent relationship between clinical rating scale scores, postural stability indices, limits of stability scores, and step and stride length variability in individuals with FA.

Multiple Sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS). As part of this disorder the myelin sheath undergoes degeneration, leading to alterations in the conductivity of axons, and impaired function. The onset of the disease occurs in young adults and clinical pathology is characterised by varying severity. These include i) Relapsing Remitting MS (RR-MS), ii) Secondary Progressive MS (SP-MS) and iii) Primary Progressive MS (PP-MS). MS is more prevalent in women and accounts for more than two thirds of all MS sufferers. MS is considered to be a multifactorial disorder with both genetic and environmental components. The prevalence of MS is dependent on geographical localisation, with lower sunlight exposure linked to higher prevalence. Also, studies show an increased risk in close relatives, or in identical twins, indicating a significant genetic component to the disorder. There are a number of genes that may plausibly be involved in MS pathophysiology. These include myelin-related genes, such as the myelin basic protein (MBP), immune-related genes, such FC receptor and osteopontin, and heat shock proteins such as xb crystallin. These candidate genes have been implicated in a variety of ways but usually through immunological and/or genetic studies. One of the most consistent findings in recent years has been the association of disease with alterations in the specific major histocompatibility complex (MHC) localised to chromosome 6p21.3, and includes MHC I, II, III. Genome wide screens have permitted the identification of loci in the genome, which are associated with MS susceptibility. The number of genes involved in MS is unknown and several case-control association studies have been undertaken to reveal the involvement of potential candidate genes. In general terms, current research is aimed at determining allelic variation of candidate genes. Such genes have been implicated in MS because they reside within susceptible regions of the chromosome associated with MS or they have a plausible potential pathophysiological role in MS. Candidate loci investigated in this study, for association with MS susceptibility, include members of the nitric oxide synthase family of metabolic proteins (inducible NOS, iNOS/NOS2A and neuronal NOS, nNOS), methylenetetrahydrofolate reductase (MTHFR), catechol-O-methyl transferase (COMT), and vitamin D receptor (VDR). The MS population used in all studies consisted of over 100 MS cases and gender, age and ethnicity matched controls. In our study of inducible and neuronal NOS genes, PCR based assays were developed to amplify a region of both promoters that contained known microsatellite variation. Supporting phyisological data suggests that the neuroinflammatory aspects of MS are associated with aberrant NO production, which may be due to aberrant regulation of NOS activity. Specific amplified products were identified by fluorescent capillary electrophoresis and allele frequencies were statistically compared using chi-squared analysis. In the nNOS and iNOS study, no association was identified with allele frequency variation and MS susceptibility (nNOS: ?2=5.63, P=0.962; iNOS: ?2=3.4; P=0.082). Similarly, no differences in allele frequencies were observed for gender or clinical course for both markers (Pvalue greater than 0.05). In short, results from this study indicate that the NOS promoter variations studied do not play a significant role in determining susceptibility to MS in the tested population. The COMT and MTHFR genes are localised at 22q12-13 and 1p36.3 respectively, regions of the genome that have been found to be positively associated with MS susceptibility. In our research, we set out to examine the G158A change in the 4th exon of the COMT gene. This functional mutation leads to an amino acid change (valine to methionine) that is directly associated with changes in the activity of COMT. The MTHFR enzyme plays a role in folate metabolism, and can be implicated in the turnover of homocysteine. Previous investigations have shown that high levels of homocysteine are encountered in MS patients, where it is also linked to demyelination in the CNS. In our study the aim was to examine the C677T variation (alanine to valine amino acid change) in the exon 4 coding region of the MTHFR gene and the G158A variation in the COMT gene. Restriction fragment length polymorphism (RFLP) analysis and gel electrophoresis was used to identify specific alleles for both COMT and MTHFR. However, as with the NOS study, no specific association was identified between MS susceptibility and variation for either of the tested COMT or MTHFR (Pvalue greater than 0.05) variants. In a final genomic investigation of the MS population, three variations in the VDR gene were analysed for association with MS susceptibility and pathology. Using RFLP analysis, three VDR variants were investigated with genotypes detected using the Taq I, Apa I and Fok I restriction enzymes. In contrast to previous genotypic analyses, this study did show a positive association, specifically between the functional variation in exon 9 of the VDR gene and MS (Taq I, 2= 7.22, P= 0.0072). Interestingly, the Apa I variant of VDR was also found to be associated with MS ( 2=4.2, P=0.04). The Taq I and Apa I variants were also found to be in very strong and significant linkage disequilibrium (D'=0.96, Pvalue less than 0.0001) and their associations were more prominent with the progressive forms of MS (SP-MS and PP-MS). In addition to genotypic analysis of a clinical population, additional research was undertaken to identify novel targets for MS susceptibility studies. Global gene expression analysis was undertaken using comparative subtractive fluorescent microarray technology to examine differences in gene activity (expression) in age and sex matched MS plaque tissue and anatomically matched normal white matter (NWM). MS plaques were obtained post mortem from MS sufferers with no drug history in the last two months before death and matched anatomically to healthy white matter from donors with no previous neurological disorders. Target arrays consisted of 5000 cDNAs and analysis was conducted using the Affymetrix 428 scanner. In this way, 139 genes were shown to be differentially regulated in MS plaque tissue compared to NWM. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue less than 0.0001, a=0.73); while 70 transcripts were uniquely differentially expressed ( 1.5-fold) in either acute or chronic active lesions. To validate the gene expression profile results, quantitative real time reverse transcriptase (RT) PCR (Q-PCR) analysis was performed. 12 genes were selected because they were shown to be differentially expressed by array analysis in this study, or because of their involvement in MS pathology. These included transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), glutathione S-transferase pi (GSTP1), crystallin, alpha-B (CRYAB), phosphomannomutase 1 (PMM1), tubulin beta-5 (TBB5), inositol 1,4,5-trisphosphate 3-kinase B (ITPKB), calpain 1 (CAPNS1), osteopontin (SPP1 or OPN), as well as the signal transducer and activator of transcription 1 (STAT1) and protein inhibitor of activated STAT1 (PIAS1). Both absolute (copy number) and comparative differences in the relative levels of expression in MS lesions and NWM were determined for each gene. The results from this study revealed a significant correlation of real time PCR results with the microarray data, while a significant correlation was also found between comparative and absolute determinations of fold. As with the results of array analysis, a significant difference in gene expression patterning was identified between chronic active and acute plaque pathologies. For example, a up to 50-fold increase in SPP1 and ITPKB levels in acute plaques contrasted with the 5-fold or less increase in chronic active plaques (P less than 0.0.1, unpaired t-Test). Of particular note, gamma-amino butyric acid receptor ?2 (GABG2), integrin ?5 (ITGB5), complement component 4B (C4B), parathyroid hormone receptor 1 (PTHR1) were found up-regulated in MS and glial derived neurotropic factor ?2 (GDNFA2), insulin receptor (INSR), thyroid hormone receptor ZAKI4 (ZAKI4) were found down-regulated in MS. Data also revealed a decreased expression of the immune related genes STAT1 and PIAS1 in acute plaques. In conclusion, this research used both genomic analysis and technologies in gene expression to investigate both known and novel markers of MS pathology and susceptibility. The study developed tools that may be used for further investigation of clinical pathology in MS and have provided interesting initial expression data to further investigate the genes that play a role in MS development and progression.

Several functions have been attributed to the cellular prion protein, PrPc, amongst which its anti-oxidant role has rapidly been gaining interest in the recent years. We and others have previously shown, that PrPc expressing cells, of neuroblastoma or epithelial origin, seem to exhibit a higher overall viability towards paraquat toxicity than cells expressing basal or low levels of the protein. Although several studies propose a protective mechanism that involves PrPc dependent activation of the superoxide dismutase (SOD) enzymatic machinery or an activation of its own intrinsic antioxidant function, others argue against this SOD-like role. Our objective was to investigate, at a biological and proteomic level, by which potential mechanism PrPc could protect neuroblastoma cells against paraquat induced oxidative damage. Using a biological aproach, we firstly evaluated the status of the Cu/Zn-SOD enzyme in Human neuroblastoma cells expressing different forms of PrPc following their exposure to paraquat. Next, we performed a proteomic study to investigate by which other potential mechanism(s), PrPc could protect the cell against paraquat induced oxidative stress. Our proteomic approach made use of an optimised two-dimensional liquid chromatography system, the ProteomeLab PF-2D, and reverse phase chromatography coupled with lava purple stained SDS-PAGE, both interfaced with tandem mass spectrometry. An interesting aspect of our study has been the development of an original immunoproteomic technique called immuno-PF2D-MS/MS, coupling classical immunological methods to a two-dimensional liquid chromatography proteomic tool interfaced with tandem mass spectrometry. We have proposed this technique for antigenic and serological characterization that have important implications in the study of biomarkers. Another important aspect of our study has been the detection of several candidates that could participate in PrPc-mediated protection against paraquat induced oxidative stress. Although, it was out of our scope to investigate each of these candidates in the present study, it presents an interesting perspective for future studies. We have, however, shown the implication of one such candidate: PARP-1. Complimentary tests will be necessary in the future to confirm the actual interaction of this candidate with PrPc.

Spinal Muscular Atrophy (SMA) is a neuronal degenerative disease caused by the mutation or loss of the Survival Motor Neuron (SMN) gene. The cause for the specific motor neuron susceptibility in SMA has not been identified. The high axonal transport/localization demand on motor neurons may be one potentially disrupted function, more specific to these cells. We therefore used a large-scale immunoprecipitation (IP) experiment, to identify potential interactors of SMN involved in neuronal transport and localization of mRNA targets. We identified KH-type splicing regulatory protein (KSRP), a multifunctional RNA-binding protein that has been implicated in transcriptional regulation, neuro-specific alternative splicing, and mRNA decay. KSRP is closely related to chick zipcode-binding protein 2 and rat MARTA1, proteins involved in neuronal transport/localization of beta-actin and microtubule-associated protein 2 mRNAs, respectively. We demonstrated that KSRP is arginine methylated, a novel SMN interactor (specifically with the SMN Tudor domain; and not with SMA causing mutants). We also found this protein to be misregulated in the absence of SMN, resulting in increased mRNA stability of KSRP mRNA target, p21cip/waf1. A role for SMN as an axonal chaperone of methylated RBPs could thus be key in SMA pathophysiology.

Prion diseases are fatal neurodegenerative disorders of humans and animals. The prion hypothesis states that PrPSc, a misfolded conformational isoform of the cellular prion protein (PrPC), is the sole component of the infectious particle. Many open questions exist in prion biology including the cellular role of PrPC, the potential involvement of auxiliary factors in prion replication, and the mechanism of PrPSc-induced toxicity in prion disease. The identification of novel prion-like proteins and authentic in vivo prion protein-interacting proteins would certainly assist the process of demystifying these unsolved mysteries. Accordingly, two newly-identified proteins with potential relevance to prion protein biology, Shadoo and DPPX, were selected for biochemical and functional characterization. Shadoo, a hypothetical prion-like protein, is revealed as being a glycoprotein which possesses many overlapping properties with PrPC including neuronal expression, C1-like endoproteolytic processing, and the ability to protect against apoptotic stimuli in cerebellar neurons. Shadoo loosely resembles the disordered N-terminal domain of PrPC and consistent with this notion, Shadoo appears to lack a well-defined structure. Remarkably, Shadoo levels in the brains of mice with clinical prion disease are significantly decreased suggesting that Shadoo may be inherently linked to prion replication or prion disease pathogenesis. These experiments define Shadoo as the third member of the prion protein family and, because of its functional similarities to PrPC, Shadoo may be a useful tool for deciphering the in vivo function of PrPC. DPPX, a neuronal type II transmembrane protein, is demonstrated to be the first protein capable of interacting with all three members of the prion protein family (PrPC, Doppel, and Shadoo) in vivo. Complex formation between prion proteins and DPPX appears to be mediated by multiple binding sites. When coupled with high levels of DPPX expression in cerebellar granular neurons, DPPX is a strong candidate for mediating phenotypic interactions between prion proteins in cerebellar cells. Thus, Shadoo and DPPX comprise two new entry points for studying prion proteins. Further investigation of the roles of Shadoo and DPPX in both the cell biology of prion proteins and prion disease may yield important clues to these enigmatic topics.

Prion diseases are fatal neurodegenerative disorders of humans and animals. The prion hypothesis states that PrPSc, a misfolded conformational isoform of the cellular prion protein (PrPC), is the sole component of the infectious particle. Many open questions exist in prion biology including the cellular role of PrPC, the potential involvement of auxiliary factors in prion replication, and the mechanism of PrPSc-induced toxicity in prion disease. The identification of novel prion-like proteins and authentic in vivo prion protein-interacting proteins would certainly assist the process of demystifying these unsolved mysteries. Accordingly, two newly-identified proteins with potential relevance to prion protein biology, Shadoo and DPPX, were selected for biochemical and functional characterization. Shadoo, a hypothetical prion-like protein, is revealed as being a glycoprotein which possesses many overlapping properties with PrPC including neuronal expression, C1-like endoproteolytic processing, and the ability to protect against apoptotic stimuli in cerebellar neurons. Shadoo loosely resembles the disordered N-terminal domain of PrPC and consistent with this notion, Shadoo appears to lack a well-defined structure. Remarkably, Shadoo levels in the brains of mice with clinical prion disease are significantly decreased suggesting that Shadoo may be inherently linked to prion replication or prion disease pathogenesis. These experiments define Shadoo as the third member of the prion protein family and, because of its functional similarities to PrPC, Shadoo may be a useful tool for deciphering the in vivo function of PrPC. DPPX, a neuronal type II transmembrane protein, is demonstrated to be the first protein capable of interacting with all three members of the prion protein family (PrPC, Doppel, and Shadoo) in vivo. Complex formation between prion proteins and DPPX appears to be mediated by multiple binding sites. When coupled with high levels of DPPX expression in cerebellar granular neurons, DPPX is a strong candidate for mediating phenotypic interactions between prion proteins in cerebellar cells. Thus, Shadoo and DPPX comprise two new entry points for studying prion proteins. Further investigation of the roles of Shadoo and DPPX in both the cell biology of prion proteins and prion disease may yield important clues to these enigmatic topics.

Genome-wide association studies (GWAS)s provide an unbiased means of exploring the landscape of complex genetic disease. As such, these studies have identified genetic variants that are robustly associated with a multitude of conditions. I hypothesize that these genetic variants serve as excellent tools for evaluation of the genetic interface between epidemiologically related conditions. Herein, I test the association between SNPs associated with either (i) plasma lipids, (ii) rheumatoid arthritis (RA) or (iii) diabetes mellitus (DM) and late-onset Alzheimer’s disease (AD) to identify shared genetic variants. Regarding the most significantly AD-associated variants, I have also attempted to elucidate their molecular function. Only cholesterol-associated SNPs, as a group, are significantly associated with AD. This association remains after excluding APOE SNPs and suggests that peripheral and or central cholesterol metabolism contribute to AD risk. The general lack of association between RA-associated SNPs and AD is also significant in that these data challenge the hypothesis that genetic variants that increase risk of RA confer protection against AD. Functional studies of variants exhibiting novel associations with AD reveal that the lipid-associated SNP rs3846662 modulates HMGCR exon 13 splicing differentially in different cell types. Although less clear, trends were also observed between the RA-associated rs2837960 and the expression of several BACE2 isoforms, and between the DM-associated rs7804356 and expression of a rare SKAP2 isoform, respectively. In conclusion, the overlap of lipid-, RA- or DM-associated SNPs with AD is modest but in several instances significant. Continued analysis of the interface between GWAS of separate conditions will likely facilitate novel associations missed by conventional GWAS. Furthermore, the identification of functional variants associated with multiple conditions should provide insight into novel mechanisms of disease and may lead to the identification of new therapeutic targets in an era of personalized genomic medicine.

Spinal Muscular Atrophy (SMA) is a neuronal degenerative disease caused by the mutation or loss of the Survival Motor Neuron (SMN) gene. The cause for the specific motor neuron susceptibility in SMA has not been identified. The high axonal transport/localization demand on motor neurons may be one potentially disrupted function, more specific to these cells. We therefore used a large-scale immunoprecipitation (IP) experiment, to identify potential interactors of SMN involved in neuronal transport and localization of mRNA targets. We identified KH-type splicing regulatory protein (KSRP), a multifunctional RNA-binding protein that has been implicated in transcriptional regulation, neuro-specific alternative splicing, and mRNA decay. KSRP is closely related to chick zipcode-binding protein 2 and rat MARTA1, proteins involved in neuronal transport/localization of beta-actin and microtubule-associated protein 2 mRNAs, respectively. We demonstrated that KSRP is arginine methylated, a novel SMN interactor (specifically with the SMN Tudor domain; and not with SMA causing mutants). We also found this protein to be misregulated in the absence of SMN, resulting in increased mRNA stability of KSRP mRNA target, p21cip/waf1. A role for SMN as an axonal chaperone of methylated RBPs could thus be key in SMA pathophysiology.

Parkinson’s disease (PD) is a progressive, degenerative, neurological disease. The progressive disability associated with PD results in substantial burdens for those with the condition, their families and society in terms of increased health resource use, earnings loss of affected individuals and family caregivers, poorer quality of life, caregiver burden, disrupted family relationships, decreased social and leisure activities, and deteriorating emotional well-being. Currently, no cure is available and the efficacy of available treatments, such as medication and surgical interventions, decreases with longer duration of the disease. Whilst the cause of PD is unknown, genetic and environmental factors are believed to contribute to its aetiology. Descriptive and analytical epidemiological studies have been conducted in a number of countries in an effort to elucidate the cause, or causes, of PD. Rural residency, farming, well water consumption, pesticide exposure, metals and solvents have been implicated as potential risk factors for PD in some previous epidemiological studies. However, there is substantial disagreement between the results of existing studies. Therefore, the role of environmental exposures in the aetiology of PD remains unclear. The main component of this thesis consists of a case-control study that assessed the contribution of environmental exposures to the risk of developing PD. An existing, previously unanalysed, dataset from a local case-control study was analysed to inform the design of the new case-control study. The analysis results suggested that regular exposure to pesticides and head injury were important risk factors for PD. However, due to the substantial limitations of this existing study, further confirmation of these results was desirable with a more robustly designed epidemiological study. A new exposure measurement instrument (a structured interviewer-delivered questionnaire) was developed for the new case-control study to obtain data on demographic, lifestyle, environmental and medical factors. Prior to its use in the case-control study, the questionnaire was assessed for test-retest repeatability in a series of 32 PD cases and 29 healthy sex-, age- and residential suburb-matched electoral roll controls. High repeatability was demonstrated for lifestyle exposures, such as smoking and coffee/tea consumption (kappas 0.70-1.00). The majority of environmental exposures, including use of pesticides, solvents and exposure to metal dusts and fumes, also showed high repeatability (kappas >0.78). A consecutive series of 163 PD case participants was recruited from a neurology clinic in Brisbane. One hundred and fifty-one (151) control participants were randomly selected from the Australian Commonwealth Electoral Roll and individually matched to the PD cases on age (± 2 years), sex and current residential suburb. Participants ranged in age from 40-89 years (mean age 67 years). Exposure data were collected in face-to-face interviews. Odds ratios and 95% confidence intervals were calculated using conditional logistic regression for matched sets in SAS version 9.1. Consistent with previous studies, ever having been a regular smoker or coffee drinker was inversely associated with PD with dose-response relationships evident for packyears smoked and number of cups of coffee drunk per day. Passive smoking from ever having lived with a smoker or worked in a smoky workplace was also inversely related to PD. Ever having been a regular tea drinker was associated with decreased odds of PD. Hobby gardening was inversely associated with PD. However, use of fungicides in the home garden or occupationally was associated with increased odds of PD. Exposure to welding fumes, cleaning solvents, or thinners occupationally was associated with increased odds of PD. Ever having resided in a rural or remote area was inversely associated with PD. Ever having resided on a farm was only associated with moderately increased odds of PD. Whilst the current study’s results suggest that environmental exposures on their own are only modest contributors to overall PD risk, the possibility that interaction with genetic factors may additively or synergistically increase risk should be considered. The results of this research support the theory that PD has a multifactorial aetiology and that environmental exposures are some of a number of factors to contribute to PD risk. There was also evidence of interaction between some factors (eg smoking and welding) to moderate PD risk.

There has been much interest in the use of therapeutics based on ribonucleic acid interference(RNAi) to inhibit synthesis of mutant proteins ever since Elbashir et al. (Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411, 494-498.) found that synthetic double stranded small interfering ribonucleic acids (siRNAs) can initiate this evolutionarily conserved process in mammalian cells. Since RNAi is able to target single genes and therefore mitigate the underlying molecular pathology of diseases, RNAi-based therapeutics will most likely benefit monogenic neurodegenerative diseases such as Huntington’s disease. It is however particularly difficult to deliver exogenous materials such as siRNAs into neurons in vivo as the blood-brain barrier (BBB) isolates the brain from the vascular system and prevents permeation of most materials. Neurons also do not take up exogenous materials readily. Therefore, effective delivery of siRNAs into the brain remains one of the biggest challenges impeding their use as a potential neurotherapeutic. Layered double hydroxide (LDH) nanoparticles are a class of anionic clay materials that have demonstrated great potential as a DNA (deoxyribonucleic acid) delivery system for a variety of mammalian cell lines due to their unique physiochemical properties. This thesis examined the feasibility of LDH as a siRNA delivery system for cultured neurons and demonstrated that the delivered siRNAs are able to effectively down-regulate synthesis of a target protein with minimal toxicity. Experiments were conducted using double stranded DNAs (dsDNAs) initially, and siRNAs were then used to verify these results. It was shown that nucleic acids(dsDNAs and siRNAs) could successfully intercalate into pristine LDHs to form nucleic acid-LDH complexes that had properties suitable for use as a delivery system in mammalian cells. These studies established that LDHs and nucleic acid-LDH complexes were biocompatible with neurons isolated from embryonic day 17.5 mouse cerebral cortex, suggesting that LDH can be used for nucleic acid delivery into cultured neurons. LDHs were also shown to successfully deliver nucleic acids into a non-neural mammalian cell line (NIH 3T3 cells). Finally, this thesis demonstrated for the first time that LDHs were able to deliver siRNAs into neurons, providing encouraging preliminary evidence that sequence specific gene silencing of the Mus Musculus Deleted in Colorectal Cancer (DCC) gene had occurred. However, down-regulation of the DCC protein did not occur consistently, suggesting that further optimisation is needed to improve the efficacy of siRNA-LDH complexes to inhibit expression of target protein in neurons. In future, LDHs should be further developed as an efficient siRNA delivery system for therapeutic gene silencing in the central nervous system using a neurodegenerative disease model such as the Huntington’s disease mouse model, which closely phenocopies the human disease. This model will allow the in vivo efficacy of these nanoparticles to be tested and subsequently improved in order to deliver siRNAs locally and systematically into the brain.