Novel Therapeutic Targets and Biomarkers for the Treatment of Progressive Supranuclear Palsy

Progressive supranuclear palsy (PSP) is a sporadic parkinsonism tauopathy characterised by the deposition of aggregations of abnormal, hyperphosphorylated four-repeat tau (4R-tau). A revised clinical diagnostic criterion for PSP allows early presentations for the full spectrum of clinical phenotypes to be recognised enabling doctors to make a more accurate diagnosis. The major genetic risk factor for sporadic PSP is a common variant in the gene encoding microtubule-associated protein tau (MAPT). Research into the biochemical and pathological pathways of tau is vital to improve the chances of developing an effective diagnostic biomarker to monitor tau pathogenesis. Neuroimaging biomarkers, such as tau PET ligands, are proving the most successful tool in providing a differential diagnosis between neurodegenerative disorders. There are currently no effective treatments for PSP, however tau-directed therapies in the last five years have rapidly advanced. Latest tau therapies are proposed to have disease-modifying effects by reducing toxic aggregations of tau through manipulating tau gene expression. After encouraging results from long awaited trials, additional funding is being injected into this field and with new results expected, this proves an exciting area for scientific discovery. This paper reviews advances in pathophysiology, diagnosis, biomarkers and disease-modifying therapeutic treatments for PSP.


Introduction
Every hour two people are diagnosed with Parkinson's (PD) in the UK. 1 Parkinson's is a heterogenous, progressive neurodegenerative disorder involving a plethora of molecular pathways and associated with variable motor and nonmotor characteristics. 1 Parkinsonism encompasses a multitude of clinical syndromes characterised by profound movement difficulties that share similar symptoms to PD. 2 Currently there are estimated to be ten million people worldwide living with PD. 3 85% of these victims have primary parkinsonism or idiopathic Parkinson's disease (PD). 1 The remaining have secondary parkinsonism or atypical parkinsonism (APS) which encapsulate vascular parkinsonism, drug induced parkinsonism, dementia with Lewy bodies (DLB) multiple system atrophy (MSA), progressive supranuclear palsy (PSP), normal pressure hydrocephalus (NSA) and corticobasal syndrome (CBS). 1 There are great difficulties in distinguishing between parkinsonism disorders, therefore research is vital to ensure accurate and early detection. Primary parkinsonism exhibits a strong response to levodopa whereas secondary parkinsonism has a weaker response. 1 For this reason, a levodopa trial may be used to discriminate between primary and secondary parkinsonism.
APS syndromes, in particular PSP, are largely under-researched solely due to the limited number of patient cases 4 compared with idiopathic PD. 5 The deficiency in research dedicated to PSP has led to frequent misunderstandings in pathology and diagnosis. However, in recent years the landscape has transformed. With more attention directed towards PSP, the original classification as an APS syndrome may not hold true, with PSP being a separate disease with a different pathological process.
Nevertheless, a sufficient understanding of PD is integral in order to distinguish between PSP and PD pathologies in an attempt to develop and modify effective treatments for PSP. In this paper both diagnostic biomarkers and therapeutic targets for PSP will be discussed. A biomarker is a characteristic that is measured as an indicator of the pathological processes associated with disease. This can be used in diagnosis and predicting progression in PSP. Therapeutic targets concern structures, molecular pathways and processes associated with PSP pathology which are treated using novel agents in an attempt to suppress pathogenesis.

Parkinson's
In 1817 Dr James Parkinson first described this debilitating disorder PD as "shaking palsy" 6 however earlier descriptions had been previously recorded. In 1872 Jean-Martin Charcot discovered two prototypes: the tremorous and the akinetic/rigid form. 7 Since then there has been a revolutionary development in understanding both clinical and pathophysiological aspects of the disease. The dramatic expansion in the clinical spectrum of parkinsonism 8 has led to numerous difficulties in developing potential drugs and therapeutic treatments. The diversity in neuropathological phenotypes threatens potential drug prospects as questions remain as to whether a drug could be applicable for more than one phenotype. This accentuates the necessity to clinically diagnose the variant phenotype, in order to guide treatment.
The diagnosis of PD is based on the presence of 1) typical signs, 2) significant and sustained response to levodopa, 9 and 3) absence of atypical features that would be suggestive of an alternative parkinsonism syndrome. 10 A range of motor and nonmotor symptoms are associated with PD. The evolution of PD takes 5-7 years during the preclinical period, but conditions worsen under symptomatic diagnosis. 11 Cardinal motor symptoms include the clinical triad bradykinesia, rigidity and tremor. 9,11,12 Nonmotor symptoms include autonomic dysfunction (constipation, sweating), neuropsychiatric (anxiety, dementia, depression), sensory (olfactory dysfunction, paraesthesia), sleep disturbance and other less common symptoms like weight loss. 9 The quality of life for PD patients is severely lowered due to depressive tendencies 13 where 5%-20% of patients have major depression and 10%-30% have minor depression. 14,15,16 Research shows that nonmotor features tend to precede motor features of PD, which are only experienced after the loss of 50% to 80% of dopaminergic neurons. 9 An estimated 80% of patients who suffer from PD twenty years after diagnosis have Parkinson's disease dementia, 17 which is closely related to Lewy body dementia. 18 In December 2019, The Guardian publicised that 26% of people with PD were initially misdiagnosed. 19 As PD presents with subtle prodromal symptoms there is overlap between PD and other APS which due to the nature of their complex disease course, makes diagnosis challenging. 20 These challenges highlight the requirement for an effective biomarker to ensure early and accurate detection of the disease and its variants. However, the invasiveness and expense of experimental procedures pose questions of the value of the proposed trial.
PD is a synucleinopathy characterised by the abnormal aggregation of α-synuclein in neurons called Lewy bodies (LBs). 21 The accumulation of abnormal α-synuclein aggregates induces the progressive degeneration of nigral dopaminergic neurons located primarily in the substantia nigra pars compacta (SNpc) found in the basal ganglia of the brain. 22 However, there is also widespread cell loss in several subcortical nuclei, including the locus coeruleus (LC). 23 Dopamine is a catecholamine neurotransmitter involved in a range of neurological processes including motor control, cognition and movement. 24 Apoptosis of dopaminergic neurons results in a fall in dopamine concentration in the striatum (the nigrostriatal pathway). [24][25][26] As a result, this disturbs the intricate balance between dopaminergic inputs and cholinergic interneuron (ChI) neurotransmission within the striatum inducing a range of the aforementioned motor symptoms. 27 New evidence suggests that tauopathy is associated with PD onset. 28,29 Tauopathies refer to heterogenous disorders characterised by abnormal tau aggregates in the brain. 29 Axons are stabilised by microtubules (MTs) which are integrated with tau proteins encoded by the axonal microtubule associated protein tau gene (MAPT) (Fig. 1). 28,30 Under pathological conditions tau proteins are post-translationally modified by abnormal hyperphosphorylation primarily at threonines (pThr) or serines (pSer) 30 causing them to form filamentous neurofibrillary tangles (NFTs) which disintegrate MTs. 29-31 Hyperphosphorylated tau proteins interact with α-synuclein disrupting the microtubule network, causing aggregation and leading to the formation of LBs and consequent axonal transport malfunction. 32 The MAPT gene comprises 16 exons on single chromosome 17q21. 33 There are six main isoforms of tau expressed in the human brain due to alternative splicing of exons E2, E3 and E10 around the Nterminal region. [34][35][36] The isoforms differ in size ranging from 352 to 441 amino acids and depending on the absence or presence of two N-terminals (N1 and N2) and the three or four microtubule binding domains (3R and 4R). 34,37 The 4R tau has four microtubule binding repeat sequences due to the inclusion of E10, which is not included in 3R tau. 37 The more repeats that exist in the tau protein the stronger the binding affinity to the microtubule therefore 4R tau binds more easily and polymerises MTs. 38 There are equal ratios of 3R:4R tau in the human brain, however neurodegenerative disorders unbalance this ratio 37 for example, PSP is characterised by the accumulation of 4R tau in neuronal and glial cells. 36   The exact aetiology for the multifactorial disease PSP is still unknown, although specific genetic defects and exposure to environmental factors pose as PSPassociated risk factors (Table 3). There has been an exponential acceleration in the rate of genetic discovery for PSP with the most well-known risk factor concerning mutations in the MAPT gene. 57 Identification of additional risk loci have been founded from genome-wide association studies. 58 These studies will provide a greater understanding of PSP pathogenesis giving direction for therapeutic approaches.
The principal risk factor associated with onset of PSP is age advancement. 59 The presence of lysosomal and mitochondrial dysfunctions, due to oxidative stress and accumulation of misfolded proteins, initiates neuronal aging. 60 Older people have been exposed to oxidative stress for a longer period and so endured more genetic mutations. The loss of regulation due to genetic mutation of these genes will result in neuronal impairment and induce the PSP motor and nonmotor symptoms cascade.
The first case-control study to investigate whether exposure to environmental toxins triggers the onset for PSP was only carried out in 2016. 61 The results fail to provide a causative agent and further identification of specific toxicants remain elusive. Recently it was discovered that induced pluripotent stem cell (iPSC)-derived iNeurons with the PSP-related tau variant R406W were more sensitive to chromium (Cr) and nickel (Ni), which operate through different mechanisms to induce apoptosis. 62 Exposure to neurotoxic doses of Cr and Ni increases the phosphorylation of tau and therefore regarded as a potent risk factor for promoting tauopathy and neuronal death. 62  Whilst the primary cause of PSP still remains ambiguous, it is important to define the clinical phenotypes of PSP to guide therapeutic treatment. This paper will analyse PSP pathophysiology; explore potential therapeutic targets and biomarkers; address salient issues with regards to drug development with the ultimate goal of developing a disease-modifying therapy before severe functional disability persists.

Neurological mechanism Genetic
Age a Higher levels of lysosomal and mitochondrial dysfunctions due to oxidative stress and accumulation of misfolded proteins in older people MAPT b Mutations in MAPT lead to abnormal phosphorylation and aggregation of tau Environmental Heavy metals such as chromium and nickel c iPSC-derived iNeurons from MAPT more sensitive to cell death induced by Cr and Ni

PSP criteria
In 2017, the International Parkinson and Movement Disorder Society (MDS)endorsed PSP Study Group established a revised clinical diagnostic criterion for PSP. This transformed diagnosis by allowing recognition of several PSP phenotypes and the development for new therapeutic strategies for PSP. 53 Experiments have shown that distinguishing between vPSP is hard due to overlapping symptoms forming false positives. 55 Clinicians must exert a broader diffusion of diagnostic criteria for PSP to ensure results are consistent and therefore comparable. The lack of enrollment from patients with rare phenotypes 63 will prevent future advances in pathophysiological and mechanistic discovery.

Use of clinical scales
At present, there are two established disease-specific scales for PSP including: the PSP rating scale (PSPRS) and PSP-Quality of Life Scale. These scales act as indicative tools used to evaluate the severity and progression of the disease. 64,65 Clinicians employ these methods to facilitate experimental predictions and comparisons of PSP phenotypes. An MDS-endorsed PSP study group recently embarked on inventing the PSP-Clinical Deficits Scale (PSP-CDS). 66 The aim was to develop a scale that is equally applicable to all vPSP to measure motor clinical deficits; predict annual progression rates; compliant in clinical care and research situations and simple to use whilst upholding robust clinimetrics. 66

Comparison of PSP clinical phenotypes
PSP phenotypes can be defined by physical characteristics (Table 2) and numerical data ( Table 4). The oldest age of onset for disease is PSP-PGF and PSP-F compared with other clinical variants. PSP-P has the longest disease duration of 6.6 years. A p<0.001 suggests statistical significance. 69 This is typical for PSP-P as early in the disease course the variant is categorised by the presence of non-PSP clinical features. 69 The highest rate of dementia was noted in PSP-RS patients (47.8%) compared with PSP-PGF with the lowest rate. 70 Therefore, data in table 4 suggests that PSP-RS is the most severe phenotype whilst PSP-PGF is one of the most benign, findings that are echoed in the literature. 53

Tau
The pathology of PSP concerns the microtubule-associated protein, tau which is encoded by MAPT. 57,72 Tau pathology is strongly associated with interference of microtubule function. 73 Since the discovery of 4R-tau deposits in clinically diagnosed patients with PSP, the attention to tau-based therapies has been intensified.

Tau function
The majority of tau protein is found in axons and expressed extensively in the brain. 39 Tau binds to the microtubule surface through the microtubule-binding-domain (MBD) and adjacent regions 74 providing stabilisation to the microtubules that construct neuronal axons. The intercellular binding interaction could be associated with regulation of axonal transport. 74,75 Recent trials using tau-knockout mice have unveiled novel functions of tau such as: DNA integrity, regulation of neuronal activity, neurogenesis, iron export. 74 However, the developmental role of tau remains unclear due to inherent difficulties in translating mouse brain data to human brain data. 76 The ability to assess the biochemical and pathological pathway of tau will improve the chances of developing an effective diagnostic biomarker to monitor tau pathogenesis.  1. Healthy Vs. PSP brain. MRI images of a PSP brain reveals loss of tissue from grey and white matter atrophy. The LHS shows the healthy brain and the RHS shows the diseased brain.

Hyperphosphorylation
Most studies have focussed on investigating the role of tau phosphorylation compared with other PTMs due to: 1) pathological tau is hyperphosphorylated in many tauopathies; 29,30 2) tau hyperphosphorylation affects microtubule stability; 29,30 3) phosphorylation is the most common tau PTM. 29 In a human brain there are a potential of 85 epitopes that can be phosphorylated of which 16 epitopes are phosphorylated in PSP brains compared to 10 epitopes in normal brains. 85 The phosphorylation occurs at threonines or serines in the N-and C-terminal domains of the amino acid sequence of MAPT and is mediated by GSK-3β, DYRK1A and PP2A. 76 The soluble and linear tau protein which has undergone this post-translational modification becomes insoluble and misfolded resulting in conformational changes in microtubules and formation of aggregates of tau called NFTs. 31 The discovery of NFTs was detected using antibodies modelled to target phosphorylated tau. 86 The formation of NFTs is accompanied by the formation of unique binding sites which can be targeted with small molecule probes. 87 This evidence has been exploited to construct therapeutic Researchers published that phosphorylated tau displays a positive interaction with αsynuclein which are both contained in Lewy bodies surrounded by NFTs. 86,90 However, investigations into the toxic interaction between the two proteins are on-going. 28 Scientists propose that there is a cascade reaction at synapses involving the aggregations of abnormal α-synuclein and tau which leads to inhibition of axon functionality. 91 However, repeat experiments are required to confirm this is fact.
Hyperphosphorylation results in neuroinflammation. PET studies show that brain macrophages and microglia are activated in PSP brains. 83 High levels of proinflammatory cytokines such as interleukin-1β 194 and 5-lipoxygenase enzyme 92 have been reported in PSP brains. These are released in response to neuronal damage following the formation of NFTs (Fig. 2). 84,92 The creation of a proinflammatory cytokine blocker could potentially be included in the therapeutic strategy.

Differences in pathogenesis between PSP phenotypes
The severity and distribution of pathological tau and atrophy varies in affected regions of the brain in different PSP phenotypes ( Fig. 3 and Fig. 4). PSP-RS tau pathology is estimated to begin in the pallido-luysonigral areas extending to the pontine nuclei, other basal ganglia structures, cerebellar dentate nucleus and frontal and parietal cortices. 39 It is visible that the subthalamic nucleus (STN) is the most severely affected region of the brain in PSP-RS patients (Fig. 3f). It is clear that patients with PSP-P and PSP-PGF experience less severe tau pathology than PSP-RS patient although there are overlaps in pattern and distribution of tau aggregations ( Fig. 3c and Fig. 3d).
Whitwell et al. presents neuroimaging analyses of brain atrophy using MRI and [ 18 ]flortaucipir uptake on PET across PSP variants to improve understanding of pathological differences and similarities. 69 The voxel-level analysis ( Fig. 4) shows that PSP-SL has the greatest grey and white matter loss throughout the frontal lobes.

Fig. 4 Three-dimensional MRI volume analysis (voxel-level analysis) showing grey (shown in red) and white (shown in blue) matter volume loss in different PSP phenotypes compared to controls.
Boxes contain important brain structures suffering from high levels of white and/or grey matter atrophy. Data not available for PSP-PI and PSP-OM.

Progressive Supranuclear Palsy Genetics
Familial (monogenic) PSP is defined as causative mutations in single genes transmitted by Mendelian inheritance in families (autosomal dominant or autosomal recessive). 93 Sporadic (idiopathic) PSP, which is the most common type, is associated with numerous genetic mutations in combination with environmental factors. 93 As PSP is a prototypical tauopathy, an understanding of PSP-associated alleles will help determine the molecular mechanism of tau pathology. 94

Genome-wide susceptibility loci for sporadic PSP
The largest independent genome wide association study (GWAS) for PSP to date by  The inclusion of exon 10 ( Fig. 1) in MAPT leads to production of 4-repeat isoforms for PSP. 58,102 PSP MAPT mutations are located in exon 10 and its splicing region (p.L284R, p.S285R, p.delN296, p.N296N, p.P301L, p.G303V, p.S305S, IVS10+3, and IVS10+6), except for R5L mutation. The R5L mutation was the first to be discovered and modifies the interaction of tau with tubulin and microtubules. 103

STX6
STX6 encodes protein syntaxin-6, a SNARE class protein, that is involved in intracellular protein trafficking along endoplasmic reticulum. 104 Although there is a lack of existing studies on the role of STX6 and its effect on tau metabolism in neuronal and glial cells, 101 it has been confirmed that STX6 is a risk gene. 97

MOBP
MOBP gene is found on chromosome 3p22.1 97 encodes the central nervous system (CNS) myelin structural protein which is produced by oligodendrocytes. 97,105 MOBP is highly expressed in the affected areas of the brain, particularly the brain stem and cerebellum. 105 Mutations in MOBP cause inaccurate myelin formation resulting in oligodendrocyte dysfunction and subsequent tau inclusions, a feature of PSP. 97 SNPs associated with both STX6 and MOBP incur demyelination and strong association with white matter which gives insight into the mechanistic role of tau. 97

Emerging loci associated with PSP
Recently, additional SNPs were discovered with significant association with PSP. 94,96 The highly expressed SLCO1A2 in PSP brains encodes a solute carrier organic anion transporter (SLCO) protein 39 and SLCO1A2 mutants are suggested to have a broader role in neurodegeneration. 96 The intergenic rs6687758 SNP is found near DUSP10. DUSP10 may impact the hyperactivation of p38 and Jun amino-terminal kinases (JNK) leading to uncontrolled hyperphosphorylation of tau, gliosis and synaptic deficits. 96 The function of p38 and JNK in PSP is yet to be discovered. 96 RUNX2 encodes a transcriptional factor that affects osteoblast differentiation and is shown to have associations with PSP pathogenesis. 94 TRIM11 has been speculated as a genetic modifier of clinical phenotype in PSP. 98 The microglial gene CXCR4 has been reported to be significantly upregulated in PSP. 99 Changes to gene expression of CXCR4 and associated microglial genes (CXCL12, TLR2, RALB, and CCR5) are suggested to strongly influence neuroinflammation. 99 Further analysis is required to understand the role of GWAS susceptibility loci and regulatory effects of SNPs on genes associated with PSP, in particular those affecting tau pathology, to direct therapeutic strategy.

Comparisons of Progressive Supranuclear Palsy with Related
Neurodegenerative Disorders

Differences in the clinical picture between AD, PD and PSP
In early stages of PSP, symptoms can be mistakenly diagnosed as idiopathic PD, Alzheimer's (AD) or MSA. 4 PSP is a rare disease 3 and so patient data is limited. It is expected that 4000 people in the UK are living with PSP 3 at any one time compared with 145,000 people with idiopathic PD 5 and 520,000 with AD. 107 The prevalence of PD is expected to increase by 18% and the incidence is estimated to increase by 14% or more between 2018-2025. 108 In 2025 it is expected that 0.32% of the total population of individuals aged 20 and above in the UK will have PD. 108 APS has a more distinct degenerative pathway, aggressive clinical presentations and clinical deterioration is faster compared with PD. 109 Table 6 summarises comparative features between neurodegenerative diseases AD, PD and PSP highlighting clear similarities and differences. The mean age of motor symptom onset for early-onset PSP (EOPSP) and PD is around 7 to 20 years younger than early-onset AD. There is a similar trend for late-onset which develops around 2-10 years later in AD compared to late-onset PSP (LOPSP) and PD. There is a shorter disease duration across all three diseases in late-onset compared to early-onset.
However, this could be confounded by the fact that older patients are more vulnerable to other age-dependent comorbidities such as heart and respiratory disease.
Despite PD and PSP sharing more similar clinical features (Table 6), PSP and AD share the same main characteristic protein accumulation, tau. PSP and AD can be separated based on burden of tau-positive lesions and severity and distribution of atrophy in the brain (Fig. 5).

Differences in brain pathology between AD and PSP
Baseline volumetric MRI measurements demonstrated varying levels of atrophy in brains affected by different neurodegenerative diseases (Fig. 5a). Fig. 5A shows that minimal grey matter atrophy exists in PSP brains compared with AD patients. The MRI shows that grey matter atrophy primarily concerns temporal and parietal lobes for AD and white matter atrophy primarily concerns midbrain pons for PSP. For PSP brains, white matter atrophy is predominantly confined to the superior surface of pons which is where the superior cerebellar peduncle emerges (Fig. 5a). Nerves

Potential Therapeutic Biomarkers for Progressive Supranuclear Palsy
Biomarkers are needed to confirm diagnosis; to monitor progression and to delineate subtypes of PSP to eliminate difficulties confronted at the prodromal stages of the disease. Pathognomonic features alone are highly unreliable due to clinical heterogeneity between PD and APS phenotypes. 125 Parkinsonism can be divided into two types of neuropathologies: α-synucleinopathies (PD, MSA and DLB) and tauopathies (PSP and CBS). 126,127 α-synucleinopathies are characterised by αsynuclein in its stable unfolded oligomer state which is the main protein component of Lewy bodies and Lewy neurites and a significant hallmark of PD. 126,127,128 Tauopathies are associated with intracellular deposition of abnormally phosphorylated tau that forms NFTs. 31, 126,127 There is considerable overlap between synucleinopathies and tauopathies potentiating that a spectrum of neurodegenerative disorders could be alleviated by therapeutic strategies that target common processes of tau and αsynuclein aggregation. 129 At present, disease-specific therapy for an individual APS does not exist. 130 As APS disorders are rare, the suitability and enrollment of patients for clinical trials is limited and therefore studies tend to assess patients with varying APS disorders to increase the size of cohorts. This makes the results less precise because variant APS disorders respond differently to treatment. 131 There is more research concerning potential AD or PD biomarkers due to higher incidence rates 1,107 compared with PSP, 3 however both are vital for accurate differentiation of the disorders.
To evaluate plausibility and accelerate the prodromal differentiation of PSP from APS or PD, longitudinal cohort studies assessing multiple therapeutic biomarkers with a significant number of diverse patients are required. 127 Using a basket trial approach and precision medicine, one can determine whether specific therapies are safer in one neurodegenerative disease than another. However, most studies have less PSP participants than PD participants. 126,[131][132][133][134][135] The size of the study required to have a chance of securing a sufficient number of PSP participants should be at least 300 for a PD or APS study. The clinical trial online platform shows that patient cohort sizes for PSP ranges from as little as 10 patients 136 to as large as 490 patients. 137 A larger cohort may increase reliability as it gives a more accurate mean allowing obvious outliers to be identified. However, if a study accepts probable and possible PSP participants, a high chance of misdiagnosis persists which invalidates the results. Furthermore, it takes time for trials to complete and to verify data with previous research to allow PSP pathogenesis to be sufficiently understood.
How is it possible to compare the validity and accuracy of measurements between different therapeutic strategies? A biomolecular algorithm or mechanism to assess the comparability between methods and tracer and between fluid-and imaging-derived biomarkers could be considered. 138 Recently a PSP genetic risk score (GRS) displayed a significant difference between EOPSP and LOPSP which proves promising for future diagnostic algorithms. 113 A medical algorithm e.g. a flowchart or table, helps improve and standardise decisions for treatment. By using an algorithm to determine whether a patient has EOPSP or LOPSP will aid in selecting the most suited treatment for the individual which is crucial for slowing disease progression.
It has become increasingly recognised that attention must digress from the most universally expressed aggregated proteins to acquire a comprehensive understanding of neurodegenerative pathology.

Neuroimaging
Currently neuroimaging measurements are considered supportive features in the MDS-PSP criteria 53 as they provide only predictive values insufficient for complete diagnosis. Fig. 5a clearly shows the evident differences between AD and PSP brains and therefore potentially in the future this will be regarded more highly as a diagnostic tool. Analysis of neuroimages of PSP brains concern patients in late stages of the disease who have presented with a PSP-RS phenotype. 157 However, early-stage investigations are crucial to allow comparisons with other phenotypes and determine suitability as a diagnostic biomarker.

Magnetic resonance imaging (MRI) evaluates morphologic parameters associated
with neurodegenerative diseases 157 eradicating the inherent issues that are encountered when solely relying on clinical diagnostic criteria for PSP. 55 A whole-brain meta-analyses was conducted using MRI to isolate disease-related atrophy of PSP. 158 The aforementioned diagnostic criteria were supported with neuroimaging permitting a more informative judgement for PSP. 53,158 The qualitative review published by Whitwell et al. shows significant atrophy in midbrain and cerebellar peduncles present at early phases of PSP using radiological biomarkers. 159 However, a widespread whole-brain meta-analysis will optimise sensitivity and specificity by comparing disease patterns of atrophy in differential diagnosis. 158 The study separately investigates disease-associated atrophy in grey and white matter in PSP and employs two common meta-analytical algorithms to warrant doublevalidation against each other ensuing reliable comparisons of results. 158 By combining white and grey matter atrophy of PSP and PD patients, subtraction analysis confirms the midbrain atrophy specific for PSP. 158 Albrecht et al. study is the largest cohort of PSP confirmed patients to date, increasing reliability of results. 158 Recently it was discovered that the combined use of P/M ratio with cardiac 123 Imetaiodobenzylguanidine (MIBG) scintigraphy could enhance the differentiation accuracy of PD from PSP. 160 Further meta-analyses of MRI studies will open the possibility of MRI use as a more effective diagnostic tool.

Quattrone et al. introduces the magnetic resonance parkinsonism index (MRPI) for
assessing midbrain atrophy as the product of the ratio of pons to midbrain area (P/M) and middle to superior cerebellar peduncles diameter (MCPd/SCPd). 133 However, Tipton et al. could not form a conclusive distinction between PSP and other neurodegenerative diseases using measurements of the cerebellar peduncle angle (CPA). 125 MRPI is recommended for clinical use and has proven to perform better than P/M ratio to distinguish between PSP and PD patients. 161 MRI shows midbrain and superior cerebellar peduncles (SCPs) atrophy for PSP patients and pontine atrophy in MSA patients. 133 Midbrain atrophy in PSP is detected by a decrease in the P/M ratio in MRI and identified by the 'hummingbird sign'. 157,162 Recently an updated version, MRPI 2.0, which includes measurements of third ventricle diameter, exhibits higher sensitivity and specificity to differentiate PSP-P patients from PD patients. 163 There has been confirmation that MRPI, MRPI 2.0, P/M and P/M 2.0 presents an acceptable sensitivity and specificity profile offering classification between healthy controls and vPSP. 63 However, it is yet to be used in diagnosis due to the lack of repeated studies. 63 Furthermore, MRI brainstem assessments lack stringent image standardization and concurrent method criteria between clinicians limits the validity of structural parameters, indexes or ratios and comparability between measurement tools. 125,133,162

Diffusion Tensor, Diffusion Weighted, Free Water and Diffusion Kurtosis Imaging
Diffusion tensor imaging (DTI) detects white matter microstructure 139,140 in disease whereas diffusion weighted imaging (DWI) detects basal ganglia abnormalities. 132 Free water imaging used to distinguish different forms of parkinsonism demonstrates high specificity and sensitivity. 141 A quantitative assessment of several brain areas in PSP patients using diffusion kurtosis imaging identified pathological changes related to region-specific tau deposits. 142 However, there is a lack of repeat studies and an inability to compare results as different scanners are used.

Tau
In 2013, the development of positron emission tomography (PET) ligands with high binding affinity to paired helical filaments of tau in NFTs in the brain enabled noninvasive detection, quantification and visualisation. 88 There have been numerous studies involving tau-specific PET ligands including first-generation (e.g. [ 18 F]AV-1451) and second-generation compounds (e.g. [ 18 F]PI-2620). 164 Notwithstanding, whilst some of these PET ligands may express high binding affinity for tau aggregates in AD brain tissue there has been insignificant levels of interaction with neuronal and glial aggregates in PSP, 165 which questions the value of PET ligands as a diagnostic tool for PSP.
The efficacy of tau PET ligands binding to 4R tau aggregates in PSP is proving challenging. Several studies produced elusive results on the binding of tau-PET ligands to tau deposits in PSP. 143,144,166 This is due to several reasons: 1) low affinity as a diagnostic tool. 143,144,166,167 In vitro studies show that [ 18 F]PI-2620 has stronger binding affinity to tau aggregates in AD and PSP brains than [ 18 F]AV-1451 ( Fig. 6 and Fig. 7). 145 Clinical trials to evaluate the utility and pharmacokinetic properties of [ 18 F]PI-2620 in human brains are in progress. 145 Researchers are concentrating on [ 18 F]PI-2620 due to first clinical impressions portraying positive outlooks with low off-target binding compared with [ 18 F]AV-1451 ( Fig. 6 and Fig. 7). 145,168 Positive results permitted the launch of phase-II for PSP patients in March 2019. 168,169 The main challenges with in vivo tau imaging are due to expression of different tau isoforms and different patterns of deposition. 170 Consequently, a single tau PET tracer may not be compatible for the heterogenous tau deposits with variable binding affinities. The blood-brain barrier obstructs free movement of molecules due to High affinity for tau Low affinity MAO-A binding Superior affinity for tau molecular size and lipophilicity. A tau PET tracer must be suitable to cross membranes and fine-tuned to selectively target tau deposits over other proteins with analogous structures. 170 Most of the clinical studies examines PET ligands in AD patients not PSP patients 145,164,165,166,168 as AD is the most common form of dementia accounting for 50-70% of cases. 107 Despite success, greater standardisation and further crosssectional longitudinal data is required to create a single test that can accurately differentiate tau pathology in neurodegenerative brains.

Metabolic
[ 18 F]-FDG-PET has been shown to assess hypometabolism in PSP. 146 Previous studies observe hypometabolism at early disease stages and so a PET biomarker could bring an earlier diagnosis. 146 One study labelled the midbrain hypometabolism on FDG-PET scans as the 'pimple sign'. 147 This is linked to midbrain atrophy and may be valuable in distinguishing between APS. 147

SPECT HMPAO/IMP
After treatment with a tracer e.g. hexamethylpropyleneamine oxime ( 99m Tc-HMPAO) or [I123] lofetamine (IMP), perfusion single-photon emission computed tomography (SPECT) occurs which is a non-invasive analysis of pathophysiological events in the brain. 146 In various reports frontal hypoperfusion was detected using SPECT HMPAO and SPECT IMP that were consolidated in Alster et al. study but a lack of specificity delayed future advances. 146

Fluid
Diagnostic tools for PSP such as radiological biomarkers are well established whilst studies involving serum/plasma biomarkers are only recently emerging. 171

Tau
In 1995, the detection of total tau (t-tau) and phosphorylated tau (p-tau) in vivo in cerebrospinal fluid (CSF) became possible through the use of assays 172 and since then it has been at the forefront of research. 72 CSF tau exists as fragments containing N-terminal and/or mid-domain epitopes but whether concentrations of these fragments vary between different tauopathies remains unknown. 72 Increased speed of clinical deterioration of PSP was predicted with higher t-tau and lower p-tau in CSF. 148

NfL
The neurofilament light chain (NfL) in CSF is being assessed as a biomarker for differential diagnosis. 109 Disruption to axonal membrane of neurons causes NfL to be released into the CSF and blood. 153 PSP shows consistent elevated levels of NfL in CSF and blood compared with healthy controls and PD patients. 72,135,149,173 This echoes the comparative severity between APS and PD, which could refer to the individual underlying pathological pathways and speed of clinical deterioration.

YKL-40
YKL-40 is a glycoprotein expressed in astrocytes and microglia which are activated in most neurodegenerative disease. 151 Current experiments are exploiting the degradative function of microglia using small molecules or antibodies for clearance of aggregated or misfolded proteins such as tau. 150,151

NfL
A blood-based biomarker is favourable compared to the invasive lumbar puncture procedure involved in CSF-based biomarkers. 88,152,153 Results show that plasma NfL concentrations are elevated in PSP patients compared to age-match healthy individuals. 152,[174][175][176] By conducting comparative analysis, it is clear that the investigation into neurological diseases using blood-based biomarkers is more prevalent. 152,175 Although data may not be transferable or applicable to PSP or PD, further knowledge around blood-based biomarkers may ignite another avenue of therapeutic exploration.

Plasma
Blood biomarkers confer advantages such as easy accessibility of blood sampling, cost and scalability. 138,177 A trial was created to evaluate the potential diagnostic accuracy of combining plasma biomarkers (α-synuclein, total tau, p-Tau181, and Aβ42), which express the major pathologies witnessed in PD and APS. 127 The results show that all four plasma biomarkers improve differential diagnosis of PD from APS.
However, only a small number of patients with PSP participated which limits reliability and application to all PSP patients. 127 The Santiago et al. study analyses the ability of RNA blood biomarkers (α-synuclein, total tau, p-Tau181, and Aβ42) to improve differential diagnosis of PD from PSP. 134 However, it failed to reach statistical significance which eradicated the potential to produce a robust biomarker. 134 Investigation into whether young plasma transfusions administered to aging mice restore the levels of regenerative agents that can rejuvenate neuron growth and cognitive function, show positive development. 180 Trials are yet to commence in patients with PSP. 156

Eye movements
A defining feature in primarily PSP-RS but also other phenotypes, is vertical supranuclear gaze palsy (SGP). 53 This is associated with decreases in vertical saccade velocity compared with horizonal saccades and decrease in gain. 50 The downward vertical SGP is only presented in the MDS-PSP criteria as suggestive of PSP. 53 Infrared and video oculography reveals that deterioration in oculomotor performance is associated with more severely damaged disease-specific brain areas and disrupted functional connections. 122

Retinal thickness
A study examining retinal nerve fibre layer (RNFL) thickness by optical coherence tomography (OCT) and scanning laser polarimetry (SLP) reveals that thinning of RNFL is more profound in PSP patients than PD patients. 181 A recent study concludes that thinning of RNFL is greater in PSP patients than PD patients who have had symptoms for more than three years. 155 OCT and SLP could be implemented in PSP diagnosis to provide further differential utility between parkinsonism syndromes.

Sebum
Interestingly, a wife of a PD patient reported that she could detect the disease by a person's odour. 156 This led to the discovery of sebum obtained from the neck region, which is likely to be the source of the distinct smell. 156 Analysis of the sebum revealed that there were four compounds with significantly different quantities compared with controls. 156 This potential non-invasive metabolomic marker could offer large scale production. 182 PSP patients are yet to be investigated for a distinct sebum smell which could distinguish PD and PSP patients.

Therapeutic Targets for Progressive Supranuclear Palsy
Currently there are various therapeutic agents under review as potential treatments for PSP (Table 8). Those that are successful inhibit pathogenic processes in an attempt to stem the progression of the debilitating disease.

Reduction of abnormal post-translational modifications
Abnormal tau phosphorylation mediates severe complications including axonal transport dysfunction; 183 tau aggregation; 184 tau mislocalisation 185 and impairs the tau degradation pathway. 186 The main kinase involved in the pathologic tau hyperphosphorylation is glycogen synthase kinase (GSK3β). 187 Abnormal exposure of phosphatase activation domain (PAD) is associated with the activation of GSK3β, a proline-directed kinase. 183 Clinical trials of several GSK3β inhibitors such as Tideglusib and AZD0530 failed to prove significant advantage or pass safety reports. 188  Therapeutic agents with lack of data are not further discussed in paper. nd: no data available.

Microtubule stabilisers
Separation of tau from microtubules displaces normal mitochondrial function and stability resulting in defects in axonal transport and synaptic transmission. 202 One possible avenue for exploration is the use of microtubule stabilisers as a therapeutic strategy to alleviate microtubule degradation.
In 2013 Epothilone D failed to qualify on PSPRS and SEADL scales. 193 Since then there has been investigation into the use of epothilone D as an inhibitor of suspected microglial migration of α-synuclein, but further PSP-focussed studies are necessary. 194 TPI-287 is an abeo-taxane, a synthetic derivative which is able to cross the bloodbrain barrier and bind to tubulin offering stability to microtubules. 208 The results from Phase I trial of TPI-287 in 33 patients (2013) and from Phase I trial of TPI-287 in 66 patients with CBD or PSP (2014) were combined and presented at CTAD conference in 2017. 209 The results were subsequently published 124 and a summary in Table 9 highlights the important changes and differences in exploratory outcomes between the neurodegenerative disorders. The aim of the trial was to investigate the tolerability and safety of TPI-287 intravenous infusions administered to PSP, CBS and AD patients.

Alternative RNA splicing modulators
Splicing therapy can be manipulated to exclude or induce a mutation to produce nonfunctional transcript in a disease-causing gene. Designing therapies to target RNA splicing is a novel approach which is yet to be applied to candidate genes such as MAPT in PSP.

Autophagy enhancers
AZP2006 is a small molecule that stimulates macroautophagy to promote tau clearance and eliminate misfolded proteins. 202 The drug passed safety targets allowing Phase IIa study to begin with estimated completion date at the end of this year. 201

Attenuation of microglial activation and inflammation
Benfotiamine (BFT), a synthetic S-acyl thiamine derivative, 219 could be offered to activate the nuclear factor erythroid 2-related factor (Nrf2)/antioxidant response element (ARE) pathway and alleviate thiamine deficiency which is associated with PSP pathogenesis. 207 Long-term treatment of BFT shows neuroprotectivity in ADinfected murine models which initiated the design for preclinical PSP trials. 207 Additionally, neuroinflammation is associated with the proinflammatory cytokine 5lipoxygenase enzyme 92 and so 5-lipoxygenase blockers could be a potential therapy.

Current therapeutic strategies and associated problems
The immune system is a highly tractable therapeutic target. 177 The ability to target protein aggregation is critical to deliver effective neuroprotective intervention for PSP but it is yet to have shown efficacy in clinical trials. 177 Recently the proposition of the gut microbiome playing an important part in neurodevelopment has presented an entirely new opportunity for exploitation. Previous studies concerning PD have suggested that alterations of gut microbiota composition might influence gut permeability, α-synuclein aggregation and regulation of T-cell immune and inflammatory response. 220,221 It remains unknown as to how the microbiota precisely modulates brain function. 221 However, it is acknowledged that the microbiome has a symbiotic relationship with metabolic and immunologic pathways in neurodegenerative disorders. 222 The regulation of bi-directional communication of the gut-brain axis involved in immune-driven pathogenesis is now considered a potential target for therapeutic intervention.
The associated problems for therapeutic intervention for PSP concern its sporadic occurrence and methodological confusion. The lack of eligible patients suitable for trial deters hopes for developing a biomarker available for market. In previous studies there have been disorder in methodological and experimental practice which has limited the ability to form conclusive results as well as distracting clinical focus. Only recently has diagnostic criteria become more advanced and defined, empowering clinicians to strengthen their understanding in providing an earlier symptomatic diagnosis.
Furthermore, a thorough analysis of positive and negative results using a standardised model will provide clearer objectives for novel researchers to exploit.

Evidence of drug administration, efficacy and delivery
Data received from murine models is not solely transferrable to humans 76 145 This explicitly demonstrates that tau data using murine models cannot be fully applicable to humans.
The need to trial potential drugs on humans who bear different genetic and environmental exposures is vital to underpin target efficiency. Drugs are most commonly administered orally or intravenously. Oral drugs need to tolerate digestive enzymes in the stomach; drug metabolism in the liver; overcome the blood-brain barrier and withstand absorption by fat tissue to reach its specific protein target.
Intravenous (IV) medication offers direct access to the site of distribution with immediate drug onset. Similarly, IV drugs must overcome the blood-brain barrier and withstand absorption by fat tissue. Furthermore, if IV administration is too fast toxic levels of drug can result in end-organ damage.

Potential application of novel Drugs for use in combination with existing therapies
Multimodal biomarkers which combine biomarkers, imaging and clinical tests will embody a more detailed analysis. The use of protein, RNA, imaging and other clinical tests to build a diagnostic model for PSP is likely to produce refined and definitive results. It has been suggested that a complimentary approach in neuroimaging using DTI with tau-PET will increase precision and accuracy of diagnosis of PSP. 223 However, advances in neuroimaging requires longer observation times and fine tuning of instruments. 146

Proposed therapeutic direction and future implications
Recent technological investigations into tau pathology has proposed the hypothesis of prion-like propagation which revealed a new extracellular tau (eTau). 224 However, this has only been investigated in AD and yet to be studied in PSP brains. Nonetheless, this proposes a different avenue of anti-tau therapeutic approaches to explore.
Furthermore, an investigation into pharmacologic restoration of genomic architecture of tau with focus on the ability to slow tauopathy could be proposed.
Latest therapeutic developments have concerned the LC, which releases noradrenaline via widespread projections into multiple target areas. 225 The extensive connectivity concerts modulatory effects in behaviour, movement and cognition. 226

Conclusion
The intricacy of the pathogenic mechanisms underlying PSP, has many multifactorial